Vaccine composition for use in immuno-compromised populations

ABSTRACT

The invention relates to nasally-administered vaccine compositions effective against infection in immuno-compromised populations. One aspect of the invention is directed to the pediatric use of the vaccine of the invention including a vaccine effective in children against seasonal influenza virus strains. A further aspect of the invention is directed to subjects of all age groups when the composition is for pandemic use.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to nasally-administered vaccine compositionseffective against infection in immuno-compromised populations.

2. Background of the Invention

Influenza vaccines currently in general use are based on live virus orinactivated virus, and inactivated virus vaccines can be based on wholevirus, “split” virus, subunit proteins or on purified surface antigens(including haemagglutinin and neuraminidase).

The socioeconomic impact of influenza and its medical burden inimmuno-compromised subjects including the elderly has been increasinglyrecognized. Moreover, immuno-compromised individual's e.g. elderly aged≧65 years are at greater risk for hospitalization and death formseasonal influenza compared with other age groups. Further,immuno-compromised individuals have high attack rates of influenzaduring epidemic periods. Unfortunately, immuno-compromised do notrespond well to vaccinations. These subjects are found to respond toinfluenza vaccination by producing lower antibody titers to influenzahemagglutinin compared to younger adults.

The number of immuno-compromised individuals has steadily increased inthe past 3 decades as a result of the dramatic improvement in survivalrates in certain malignancies, due to increased intensity and complexityof chemotherapy regimens, the number of individuals undergoing curativeand life-saving hematopoietic stem cell transplantation and solid organtransplantation followed by immunosuppressive therapy, a dramaticdecrease in morbidity and mortality and improved quality of life inindividuals infected with human immunodeficiency virus (HIV).

Individuals with sub-optimal immune function due to disease or therapyare recognised to be at increased risk form influenza relatedcomplications. Concerns about influenza within immuno-compromisedpopulations include an impaired response to vaccination and higher riskof complicated infection with increased mortality, greater and prolongedvirus shedding with implications for control of transmission andpossible adverse effects of vaccination.

Immuno-compromised subjects include in addition to persons aged ≧65years, pregnant women, patients receiving chemotherapy, patients onimmune-suppressive drugs, such as organ transplant patients, HIVinfected individuals. A non-limiting list of what is consideredimmuno-compromised subjects is shown in table 1.

TABLE 1 Immuno-compromised subjects include the following individualsPersons aged ≧65 Pregnant women Persons with cancer Persons receivingchemotherapy Persons receiving radiation thereapy Persons undergoinghematopoietic allogenic stem cell transplantation Persons undergoinghematopoietic autologous stem cell transplantation Persons undergoingsolid organ transplants Persons with graft-versus-host disease Personswith HIV Persons receiving immunosuppressive medication e.g.glucocorticoid therapy Persons with chronic diseases e.g. end stagerenal disease, diabetes, cirrhosis

Studies have shown that conventional parenteral vaccines have decreasedability to induce satisfactory protective immunity in immuno-compromisedindividuals compared to the generally immuno-competent population.Hence, even “mild” influenza pandemics like the influenza A(H1N1)pandemic was associated with substantial mortality in the elderly andimmune-compromised.

Pregnancy is an immune-compromised state; during pregnancy, the immunesystem does not work at full capacity. Because of this, the body'simmune system in pregnancy has a harder time fighting off the influenzavirus, and the flu therefore tends to be more severe. In fact, pregnantwomen have been disproportionately affected by severe disease in allinfluenza pandemics over the past century. In the 1918 flu pandemic, forexample, half of all pregnant women with the flu experienced pneumonia.Of these, half died—resulting in an astounding and tragic death rate of25% among pregnant women who got the flu. In the 1957 pandemic, amongwomen of reproductive age, half of all reported deaths occurred inpregnant women.

During inter-pandemic periods, influenza viruses that circulate arerelated to those from the preceding epidemics. The viruses spread amongpeople with varying levels of immunity from infections earlier in life.Such circulation, in a phenomenon known as antigenic drift, over aperiod of usually 2-3 years, promotes the selection of new strains thathave changed enough to cause an epidemic again among the generalpopulation. Drift variants may have different impacts in differentcommunities, regions, countries or continents in any one year, althoughover several years their overall impact is often similar. Typicalinfluenza epidemics cause increases in incidence of pneumonia and lowerrespiratory disease as witnessed by increased rates of hospitalisationor mortality. The immuno-compromised, especially the elderly or thosewith underlying chronic diseases, are most likely to experience suchcomplications, but young infants also may suffer severe disease. In onesense young children can also be considered immune-compromised, as theirimmune system is not fully developed and does not respond as well as anadult's immune system. Infants are in their first three months of lifesusceptible to infections that are not common in older individuals (suchas Streptococcus agalactiae) and infants rely on maternal antibody forthe first few month of life. Infants do not respond to certain vaccinesin the same way as adults and are unable to produce effective antibodiesto polysaccharide antigens until around 5 years of age. The immunesystem grows and develops with the child and does not fully resemblethat of an adult until puberty, when sex hormones may be responsible forthe full maturation of the child's immune system.

At unpredictable intervals, novel influenza viruses emerge through aprocess known as “antigenic shift” and are able to cause pandemics.Antigenic shift is the process by which two or more different strains ofa virus combine to form a new subtype having a mixture of the surfaceantigens of the two or more original strains. Antigenic shift is aspecific case of reassortment or viral shift that confers a phenotypicchange. Thus, an influenza pandemic occurs when a new influenza virusappears against which the human population has no pre-existing immunity.

Antigenic shift is contrasted with antigenic drift, which is the naturalmutation over time of known strains of influenza which may lead to aloss of immunity, or in vaccine mismatch. Antigenic drift occurs in alltypes of influenza including influenza virus A, influenza B andinfluenza C. Antigenic shift, however, occurs only in influenza virus Abecause it infects more than just humans.

During a pandemic, antiviral drugs will not be sufficient or effectiveenough to cover the needs and the number of individuals at risk ofpotentially life-threating influenza disease. The development ofsuitable vaccines is essential in order to achieve protective antibodylevels in immunologically naive subjects.

These problems may be countered by adjuvantation and/or optimal vaccinedelivery the aim of which is to increase immunogenicity of the vaccinein order to be able to decrease the antigen content and thus increasethe number of vaccine doses available. The use of an adjuvant may alsohelp prime the immune system against an antigen in a population with nopre-existing immunity to the specific influenza strain. An adjuvant mayalso enhance the delivery of the vaccine and thereby decrease the amountof antigen needed to induce an immune response. The vaccine deliveryand/or the route of vaccination might be of high importance. Mostinfluenza vaccines are delivered parenterally and therefore mainlyinduce immunity against influenza in the blood. However, influenzaviruses enter our bodies through our nose or mouth i.e. through mucosalmembranes. By delivering an influenza vaccine to the nose one can induceinfluenza-specific immunity in both the mucosa and in the blood. Thismight be of benefit when aiming to induce protective immunity againstinfluenza, especially in individuals with no prior immunity to theinfluenza vaccine strain or to any influenza.

New non-live vaccines, such as a vaccine based on a whole inactivatedvirus or on part from an inactivated virus, able to induce protectiveimmunity against influenza disease in individuals with no pre-existingimmunity to the vaccine antigen are needed. Individuals withoutsufficient pre-existing immunity to influenza and/or with weakenedimmune status include immuno-compromised individuals, young children andlarge parts of the world wide population (or all) in case of a pandemic.The present invention is directed particularly to immuno-compromised,e.g. elderly. This group especially is in need of a safe, non-livevaccine that can boost an immunological response against influenza. Newvaccines that could be used as peri-pandemic vaccines to prime animmunologically naive population against a pandemic strain before orupon declaration of a pandemic are also needed. The present invention isdirected particularly to immuno-compromised individuals and notably canbe readily administered due to being formulated for nasal administrationand only containing inactivated antigens from pathogens e.g. virus orparts of viruses, thus not requiring medically trained personnel.Formulations of vaccine antigens with potent adjuvants allow forenhancing immune responses.

SUMMARY OF THE INVENTION

It is an object of the invention to provide vaccines that are able toinduce an immune response and provide protective immunity against bothseasonal and pandemic virus strains and other pathogenic organisms insubjects with an impaired immune system. One aspect of the invention isdirected to the pediatric use of the vaccine of the invention includinga vaccine effective in children against seasonal influenza virusstrains. A further aspect of the invention is directed to subjects ofall age groups when the composition is for pandemic use.

A first aspect of the invention is directed to a composition comprising

-   -   i) one or more non-live antigens, and    -   ii) an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides        for use as an intranasally administered vaccine, wherein said        vaccine is for immunization of immuno-compromised subjects.

The composition is formulated for use as an influenza vaccine forintranasal administration. The invention was developed for use as avaccine for the intranasal immunization of influenza inimmune-compromised subjects.

A second aspect of the invention is directed to a composition comprising

-   -   one or more non-live influenza virus antigens, and    -   an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides        for use as an intranasally administered vaccine to        immuno-compromised subjects.

A third aspect of the invention is directed a composition comprising

-   -   i) one or more Streptococcus pneumoniae antigens, and    -   ii) an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides        for use as an intranasally administered vaccine for use in        immune-compromised subjects for the prevention of infection with        Streptococcus pneumoniae or for reducing the severity of        symptoms associated with an infection with Streptococcus        pneumoniae.

A fourth aspect of the invention is directed to a method of immunizationof immuno-compromised subjects by intranasal administration of acomposition comprising

-   -   i) one or more non-live influenza virus antigens, and    -   ii) an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Development of HI antibody titers against H1N1 A/Ned/602/09 (A).Ferrets of group 1, 3-6 were intranasally inoculated by nasal drops ondays 0, 21 and 42 and ferrets of group 2 were subcutaneously injected ondays 21 and 42. HI antibody titers were determined in sera collectedprior to the immunizations on day 0, 21 and 42 and after the lastimmunization on days 64 and 70. Group 1 (control, i.n. saline), group 2(s.c. TIV), group 3 (i.n. Endocine™ ™ adjuvanted split antigen at 5 μgHA), group 4 (i.n. Endocine™ ™ adjuvanted split antigen at 15 μg HA),group 5 (i.n. Endocine™ ™ adjuvanted split antigen at 30 μg HA) andgroup 6 (i.n. Endocine™ ™ adjuvanted inactivated whole virus antigen at15 μg HA). Bars represent geometric mean of 6 animals per group with 95%CI (GMT+/−CI95).

FIG. 2: HI titers against distant viruses.

Ferrets of group 1, 3-6 were intranasally inoculated by nasal drops ondays 0, 21 and 42 and ferrets of group 2 were subcutaneously injected ondays 21 and 42. HI antibody titers were determined in sera collectedprior to the immunizations on day 0, 21 and 42 and after the lastimmunization on days 64 and 70. Group 1 (control, i.n. saline), group 2(s.c. TIV), group 3 (i.n. Endocine™ adjuvanted split antigen at 5 μgHA), group 4 (i.n. Endocine™ adjuvanted split antigen at 15 μg HA),group 5 (i.n. Endocine™ adjuvanted split antigen at 30 μg HA) and group6 (i.n. Endocine™ adjuvanted inactivated whole virus antigen at 15 μgHA). Bars represent geometric mean of 6 animals per group with 95% CI(GMT+/−0195). For GMT calculations, the ≦55 value was replaced with theabsolute value 5. A: Antibody titers against H1N1 A/Swine/Ned/25/80. B:Antibody titers against H1N1 A/Swine/Italy/14432/76. C: Antibody titersagainst H1N1 A/New Jersey/08/76.

FIG. 3: Development of VN antibody titers against H1N1 A/Ned/602/09.

Ferrets of group 1, 3-6 were intranasally inoculated by nasal drops ondays 0, 21 and 42 and ferrets of group 2 were subcutaneously injected ondays 21 and 42. VN antibody titers were determined in sera collectedprior to the immunizations on day 0, 21 and 42 and after the lastimmunization on days 64 and 70. Group 1 (control, i.n. saline), group 2(s.c. TIV), group 3 (i.n. Endocine™ adjuvanted split antigen at 5 μgHA), group 4 (i.n. Endocine™ adjuvanted split antigen at 15 μg HA),group 5 (i.n. Endocine™ adjuvanted split antigen at 30 μg HA) and group6 (i.n. Endocine™ adjuvanted inactivated whole virus antigen at 15 μgHA). Bars represent geometric mean of 6 animals per group with 95% CI(GMT +/−CI95).

FIG. 4: Comparison of the vaccine (Immunose™ FLU comprising 15 μg HAsplit influenza antigen with 20 mg/ml (2%) Endocine™) of the presentinvention with other adjuvanted vaccine products, FluMist (liveattenuated vaccine) and injectable vaccines in naïve ferrets.

FIG. 5 a: Shows the influenza specific IgG1 titer response over time inold mice immunized with Immunose™ Flu (circle), in old mice immunizedwithout adjuvant (square), in old mice receiving intranasal salinesolution (plus sign) and in young mice receiving Immunose™ Flu(triangle).

FIG. 5 b: Shows the influenza specific IgG2a titer response over time inold mice immunized with Immunose™ Flu (circle), in old mice immunizedwithout adjuvant (square), in old mice receiving intranasal salinesolution (plus sign) and in young mice receiving Immunose™ Flu(triangle).

FIG. 5 c: Shows the influenza specific IgA titer response over time inold mice immunized with Immunose™ Flu (circle), in old mice immunizedwithout adjuvant (square), in old mice receiving intranasal salinesolution (plus sign) and in young mice receiving Immunose™ Flu(triangle).

Table 5: Efficacy of Endocine™ formulated 2009 H1N1 vaccines in ferretsdemonstrated by clinical, virological and gross-pathology parameters.

: Group 1 (control, i.n. saline), group 2 (s.c. TIV), group 3 (i.n.Endocine™ adjuvanted split antigen at 5 μg HA), group 4 (i.n. Endocine™adjuvanted split antigen at 15 μg HA), group 5 (i.n. Endocine™adjuvanted split antigen at 30 μg HA) and group 6 (i.n. Endocine™adjuvanted inactivated whole virus antigen at 15 μg HA).

Clinical Scores. Survival, number of animals that survived up to 4 dpi;fever (° C.), maximum temperature increase presented as average withstandard deviation, number of animals in which fever was observed inparentheses, (*), body temperature of 1 animal in group 4 was notavailable due to malfunction of the recorder; % body weight loss between0 and 4 dpi presented as average with standard deviation, number ofanimals with body weight loss in parentheses. Virology. Virus sheddingin nose and throat swab samples, area under the curve (AUC) fortitration results 1-4 dpi, number of animals showing 1 or more viruspositive swab in parentheses; virus load in lung and turbinates(log₁₀TClD₅₀/g) on 4 dpi presented as average with standard deviation,or the lower limit of detection in case all animals in the group werevirus negative, number of animals with lung/turbinate virus inparentheses.

Gross pathology. % of estimated affected lung parenchyma by visualexamination during necropsy on 4 dpi presented as average with standarddeviation, number of animals with affected lung in parentheses;lung/body weight ratio (×10²) on 4 dpi presented as average withstandard deviation.

Table 6: Semi-quantitative scoring for histopathological parameters on 4dpi.

^(a): Group 1 (control, i.n. saline), group 2 (s.c. TIV), group 3 (i.n.Endocine™ adjuvanted split antigen at 5 μg HA), group 4 (i.n. Endocine™adjuvanted split antigen at 15 μg HA), group 5 (i.n. Endocine™adjuvanted split antigen at 30 μg HA) and group 6 (i.n. Endocine™adjuvanted inactivated whole virus antigen at 15 μg HA).

Histopathology. Semi-quantitative scoring for histopathologicalparameters on 4 dpi. Extent of alveolitis/alveolar damage, score: 0, 0%;1, 25%; 2, 25-50%; 3, >50%; severity of alveolitis, score: noinflammatory cells (0); few inflammatory cells (1); moderate numbers ofinflammatory cells (2); many inflammatory cells (3); alveolar oedema,alveolar haemorrhage and type II pneumocyte hyperplasia were scored aspositive slides (no=0, yes=1); All histopathology results are presentedas average with standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

In describing the embodiments of the invention specific terminology willbe resorted to for the sake of clarity. However, the invention is notintended to be limited to the specific terms so selected, and it isunderstood that each specific term includes all technical equivalentswhich operate in a similar manner to accomplish a similar purpose.

The term “immuno-compromised” means subjects aged ≧65 years and pregnantwomen. The term also covers persons of all age groups with an impairedimmune system as a result of genetic defect, pathogen inducedsuppression of the immune system or a drug induced suppression of theimmune system.

Immuno-compromised patients may therefore include, but are not limitedto the following patient classes; cancer patients, persons receivingchemotherapy, persons receiving radiation therapy, organ transplantpatients, persons undergoing solid organ transplants, stem celltransplant patients, persons undergoing hematopietic allogenic stem celltransplantation, persons undergoing hematopoietic autologous stem celltransplantation. HIV infected patients, persons with AIDS, patients withgraft-versus-host disease, patients on immune suppressive drugs e.g.glucocorticoid therapy and steroid therapy, persons with chronicdiseases e.g. end stage renal disease, diabetes, cirrhosis.

The term “peri-pandemic period” refers to the time period surrounding apandemic. Given pandemics are time periods officially identified by WHO,the invention relates to the time period immediately prior the officialrecognition of the pandemic and immediately following a pandemic, duringwhich time vaccination is recommended.

The one or more non-live influenza virus antigens in the composition ofthe invention can be from one or more influenza strain, A, B and/or Cstrain. A vaccine composition that is able to prime an immune responseand provide protective immunity against pandemic influenza strainsnormally only contains antigens from one influenza A strain (monovalent)whereas a vaccine composition that is able to prime an immune responseand provide protective immunity against seasonal influenza strainsnormally contains antigens from three or more different strains(trivalent or quadrivalent). Most commonly two different influenza Astrains and one or more influenza B strains.

The invention is further directed to a method of immunization before orduring an epidemic or pandemic period comprising intranasallyadministering a vaccine composition comprising a composition of theinvention as well as to a method of immunization of paediatricsubjection comprising intranasally administering a vaccine compositioncomprising a composition of the invention and still further directed toa method of immunization of naïve subjects comprising intranasallyadministering a vaccine composition comprising a composition of theinvention.

The invention is directed to the immuno-compromised e.g. the elderly asthis population is challenged when it comes to common vaccinestrategies. As people age numerous changes occur in the immune system.It is well established that the immune system begins to lose some of itfunctions with age and become unable to respond as quickly or asefficiently to stimuli as in the generally immune-competent adultpopulation. The changes that occur with advancing age are associatedwith significant clinical manifestations such higher incidences ofinfectious diseases (e.g. pneumonia and influenza). Both changes in thehumoral and cellular immune response occur with advancing age, much ofthe decrease in immune responsiveness seen in the elderly population isassociated with changes in the T cell response. The loss of effectiveimmune activity is largely due to alterations within the T cellcompartment which occur, in parts, as a result of thymic involution.With age people become immuno-compromised as a result ofimmunosenescence. Immunosenescence is a term used to describe reductionof immune functions in elderly aged ≧65 years old. Increasing age istherefore associated with increased susceptibility to infections andpoor response to vaccinations. For these reasons there is a need formore efficient vaccines for the elderly population such as the presentinvention.

The immuno-compromised populations have a weakened immune system. Aperson may become immuno-compromised as a result of natural courses suchas pregnancy and age or as a result of disease or the therapeutictreatment. In addition to age associated immunosenescence individualsmay become immuno-compromised as a result of diseases affecting theimmune system as well as therapeutic treatment. Individuals with chronicviral infections, such as human immunodeficiency virus (HIV) thatdirectly targets the CD4 T cells of immune system are on lifelongantiviral and immunosuppressive drugs to maintain a low virus count,which in turn leads to a weakened immune system. Other chronic viralinfections such as hepatitis B virus (HBV) and hepatitis C virus (HCV)that require prolonged treatments are also associated with animmuno-compromised state resulting in increased susceptibility withbacterial, fungal, or other viral pathogens. Organ transplantationpatients are another patient group who are classified asimmuno-compromised as they are on immunosuppressive drugs to preventthat their immune system rejects the transplanted organ. Further, somemalignancy treatments may also lead to an immuno-compromised state astreatments in addition to killing and preventing cancer growth severelyimpair the immune system.

A collective problem for the immuno-compromised individuals is that theydo not respond well to parenteral vaccines and there is therefore a needfor new approaches to increase the vaccine success rates in thispopulation. The present invention offers such a new approach.

There is a need for safe and effective vaccines against seasonalinfluenza and other opportunistic pathogens suitable for adults andchildren with immunosuppressive conditions and the elderly aged ≧65years as well as pregnant women. The immuno-compromised subjects arevulnerable to severe or complicated infections from e.g. influenza. Forexample, in the USA an estimated average 225,000 hospitalizations and36,000 deaths per annum are attributable to seasonal influenza.

Live attenuated virus vaccines are associated with safety concerns.Flumist®/Fluenz has not been approved, due to these safety issues, foruse in small children under 2 years of age, the elderly or otherwiseimmune-compromised. Paradoxically, it is the immuno-compromised subjectswhich are a particularly high risk group for influenza. Flumist® isapproved for older children but is a live attenuated virus vaccine.Further, Fluenz must not be used in people who are hypersensitive(allergic) to active substances or any of the other ingredients, togentamicin, or to eggs or egg proteins. It must also not be given topeople with weakened immune systems due to conditions such as blooddisorders, symptomatic HIV infection and cancer or as a result ofcertain medical treatments. It must also not be given to children whoare receiving treatment with salicylates (e.g. painkillers such asaspirin).

It has surprisingly been found that intranasal administration ofadjuvanted non-live influenza vaccines induced very high immuneresponses and subsequent complete protection against influenza diseasein ferrets with no pre-existing immunity to the vaccine antigen. Boththe whole and split non-live antigen vaccines gave superior results overthe injected commercially available influenza vaccine, Fluarix®.Illustrated by example 1.

The composition of the invention does not utilize a live attenuatedvirus but rather non-live influenza virus antigens. Moreover, it can beadministered intranasally. The intranasal administration of thecomposition of the invention allows for its generalized use andadministration without specialized training, such as throughout thepopulation during peri-pandemic and pandemic periods byself-administration. The use of non-live influenza virus antigens allowsfor its use in small children without the safety concerns associatedwith live attenuated virus vaccines. The inventors have developed avaccine efficacious in immuno-compromised subjects which may beintranasally administered, thereby having the above-mentioned advantagesand meeting an important need for vulnerable populations and classes ofpatients.

The invention is directed, in a first aspect, to a compositioncomprising

-   -   i) one or more non-live antigens, and    -   ii) an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides        for use as an intranasally administered vaccine, wherein said        vaccine is for immunization of immuno-compromised subjects.

The composition of the invention is suitable for use as an influenzavaccine for intranasal administration. The composition of the inventionis directed for use as a vaccine for the intranasal immunization againstinfluenza in immuno-compromised subjects. In one embodiment thecomposition is for use as a vaccine for immunization of persons aged ≧65years. In one embodiment the composition is for use as a vaccine forimmunization of pregnant women.

The influenza viruses consist of three types A, B, and C. Influenza Aviruses infect a wide variety of birds and mammals, including humans,horses, pigs, ferrets, and chickens. Influenza B is present in humans,ferrets and seals and C is present in humans, dogs and pigs. Animalsinfected with Influenza A often act as a reservoir for the influenzavirus, by generating pools of genetically and antigenically diverseviruses which are transmitted to the human population. Transmission mayoccur through close contact between humans and the infected animals, forexample, by the handling of livestock. Transmission from human to humanmay occur through close contact, or through inhalation of dropletsproduced by coughing or sneezing.

The outer surface of the influenza A virus particle consists of a lipidenvelope which contains the glycoproteins hemagglutinin (HA) andneuraminidase (NA). The HA glycoprotein is comprised of two subunits,termed HA1 and HA2. HA contains a sialic acid binding site, which bindsto sialic acid found on the outer membrane of epithelial cells of theupper and lower respiratory tract, and is absorbed into the cell viareceptor mediated endocytosis. Once inside the cell, the influenza virusparticle releases its genome, which enters the nucleus and initiatesproduction of new influenza virus particles. NA is also produced, whichcleaves sialic acid from the surface of the cell to prevent recapture ofreleased influenza virus particles. The virus incubates for a shortperiod, roughly five days in a typical case, although the incubationperiod can vary greatly. Virus is secreted approximately one day priorto the onset of the illness, and typically lasts up to three to fivedays. Typical symptoms include fever, fatigue, malaise, headache, achesand pains, coughing, and sore throat. Some symptoms may persist forseveral weeks post infection.

Different strains of influenza virus are characterized primarily bymutations in the HA and NA glycoproteins, and thus HA and NA are used toidentify viral subtypes (i.e., H5N1 indicates HA subtype 5 and NAsubtype 1). As such, influenza vaccines often target the HA and NAmolecules. Conventional influenza virus vaccines often utilize wholeinactivated viruses, which possess the appropriate HA and/or NAmolecule. Alternatively, recombinant forms of the HA and NA proteins ortheir subunits may be used as vaccines. The antigen in the vaccinecomposition may be inactivated antigens such as e.g. whole inactivatedviruses, split antigens, subunit antigens, recombinant antigens orpeptides. The term “antigen” or “immunogen” is defined as anything thatcan serve as a target for an immune response. The term also includesprotein antigens, recombinant protein components, virus like particles(VLPs) as well as genetically engineered RNA or DNA, which—when injectedinto the cells of the body—the “inner machinery” of the host cells“reads” the DNA and uses it to synthesize the pathogen's proteins.Because these proteins are recognised as foreign, when they areprocessed by the host cells and displayed on their surface, the immunesystem is alerted, which then triggers a range of immune responses. Theterm also includes material, which mimic inactivated bacteria or virusesor parts thereof. The immune response can be either cellular or humoraland be detected in systemic and/or mucosal compartments.

However, influenza is an RNA virus and is thus subject to frequentmutation, resulting in constant and permanent changes to the antigeniccomposition of the virus. The antigenic composition refers to portionsof the polypeptide which are recognized by the immune system, such asantibody binding epitopes. Small, minor changes to the antigeniccomposition are often referred to as antigenic drift. Influenza Aviruses are also capable of “swapping” genetic materials from othersubtypes in a process called reassortment, resulting in a major changeto the antigenic composition referred to as antigenic shift. Because theimmune response against the viral particles relies upon the binding ofantibodies to the HA and NA glycoproteins, frequent changes to theglycoproteins reduce the effectiveness of the immune response acquiredagainst influenza viruses over time, eventually leading to a lack ofimmunity. The ability of influenza A to undergo a rapid antigenic driftand shift can often trigger influenza epidemics due to the lack ofpre-existing immunity to the new strain.

Vaccination to prevent influenza is particularly important for personswho are at increased risk for severe complications from influenza or athigher risk for influenza-related outpatient, ED or hospital visits. TheCentre for Disease Control (CDC) recommends that in situations oflimited vaccine supply vaccination efforts should focus on deliveringvaccination to persons at risk of developing severe compilationsattributable to influenza. Persons at increased risk may include but arenot limited to all children aged 6 through 59 months;

all persons aged ≧50 years;adults and children who have chronic pulmonary (including asthma) orcardiovascular (except isolated hypertension), renal, hepatic,neurologic, hematologic, or metabolic disorders (including diabetesmellitus);persons who have immunosuppression (including immunosuppression causedby medications or by HIV infection);women who are or will be pregnant during the influenza season;children and adolescents (aged 6 months through 18 years) who arereceiving long-term aspirin therapy and who might be at risk forexperiencing Reye's syndrome after influenza virus infection;residents of nursing homes and other long-term care facilities.

The features of an influenza virus strain that give it the potential tocause a pandemic outbreak are: it contains a new haemagglutinin comparedto the haemagglutinin in the recently circulating strains, which may ormay not be accompanied by a change in neuraminidase subtype; it iscapable of being transmitted horizontally in the human population; andit is pathogenic for humans. A new haemagglutinin may be one which hasnot been evident in the human population for an extended period of time,probably a number of decades, such as H2. Or it may be a haemagglutininthat has not been circulating in the human population before, forexample H5, H9, H7 or H6 which are found in birds. In either case themajority, or at least a large proportion of, or even the entirepopulation has not previously encountered the antigen and isimmunologically naive to it.

The vaccine of the invention is particularly directed toimmuno-compromised subjects, e.g. the elderly aged ≧65 years. Theinvention is also intended for subjects with a disease or therapyinduced immuno-compromised state. In one embodiment the composition ofthe invention is for use in cancer patients. In one embodiment thecomposition is for use in pregnant women. In one embodiment thecomposition of the invention is for use persons receiving chemotherapy.In one embodiment the composition of the invention is for use personsreceiving radiation therapy. In one embodiment the composition of theinvention is for use in organ transplant patients. In one embodiment thecomposition of the invention is for use persons undergoing solid organtransplants. In one embodiment the composition of the invention is foruse stem cell transplant patients. In one embodiment the composition ofthe invention is for use persons undergoing hematopietic allogenic stemcell transplantation. In one embodiment the composition of the inventionis for use persons undergoing hematopoietic autologous stem celltransplantation. In one embodiment the composition of the invention isfor use HIV infected patients. In one embodiment the composition of theinvention is for use persons with AIDS. In one embodiment thecomposition of the invention is for use patients with graft-versus-hostdisease. In one embodiment the composition of the invention is for usepatients on immune suppressive drugs e.g. glucocorticoid therapy. In oneembodiment the composition of the invention is for use in personsreceiving steroid therapy. Further, the composition of the invention isintended, as a vaccine for immuno-compromised individuals of all agegroups during pandemic or peri-pandemic periods. In one embodiment theinvention is intended for pediatic immuno-compromised subjects.

The composition is therefore particularly directed to pediatricimmuno-compromised subject during a pandemic. The pediaticimmune-compromised subjects may be children under 18 years old, such aschildren 0 to 18 years, particularly children aged 12 and under.Typically, the children are less than 8 years of age, such as 6 yearsold or less. An important intended class of patients for the vaccine ofthe invention is particularly immuno-compromised children of 2 months toless than 9 years of age, typically immuno-compromised children of age 3months to less than 9 years old, such as of age 6 months to less than 8years old, most typically of age 6 month to less than 7 years old, suchas of age 6 months to less than 72 months, or of age 6 months to 60months or of age 6 months to 24 months. The composition of the inventionis intended, at least in part, as a vaccine for pediatric use inimmuno-compromised subjects.

The immuno-compromised subjects may be of all age groups when thecomposition is particularly directed to a vaccine for use duringpandemic or peri-pandemic periods.

Intranasal administration is intended to mean administration to the noseby any mode of administration such as by spraying the vaccine into thenasal cavity or by administering the vaccine via pipette by dripping thevaccine into the nasal cavity or onto the nasal mucosal wall.

The composition advantageously comprises one or more non-live influenzavirus antigens rather than live attenuated virus. As stated, this avoidssafety concerns both in the selection of the patient class but also interms of production, distribution and disposal. The non-live influenzavirus antigen may be selected from the group consisting of wholeinactivated virus, split virus, subunit influenza antigen andrecombinant antigens. The use of recombinant proteins can be used toincrease the titer of neutralizing antibodies produced against achallenge with the virus. The glycosylation of HA plays an importantrole in the ability of the immune response to elict an antibody responseand the virus ability to evade the immune system. Hence recombinant HAproteins can be generated containing heterogeneous complex-type glycansas well as recombinant proteins which are monoglycosylated ornon-glycosylated with increased immunogenicity.

Preferably, the non-live influenza virus antigen is a split antigen or asubunit influenza antigen, more preferably a split antigen.

The influenza A genome contains 11 genes on eight pieces of RNA,encoding for 11 proteins: hemagglutinin (HA), neuraminidase (NA),nucleoprotein (NP), M1, M2, NS1, NS2(NEP: nuclear export protein), PA,PB1 (polymerase basic 1), PB1-F2 and PB. Non-live influenza virusantigens may be selected from any one protein or combination of proteinsfrom the virus.

The composition of the invention may comprise any inactivated influenzavirus. As understood by the person skilled in the art, the influenzavirus varies from season to season and also by geographic area andpopulations in which they infect. The present invention is directed tovaccines comprising an adjuvant of the invention and non-live influenzavirus antigens from one or more influenza virus. The non-live influenzaantigen used in the vaccine composition of the invention will be anyantigenic material derived from an inactivated influenza virus. Forinstance, it may comprise inactivated whole virus particles.Alternatively, it may comprise disrupted virus (split virus) wherein forinstance an immunogenic protein, for example M2 ion channel protein, orglycoproteins are retained. Purified preparations of influenza membraneglycoproteins, haemagglutinin (HA) and/or neuraminidase (NA) may be usedas the antigenic material in the vaccine composition. A vaccinecomposition according to the invention may comprise one or more types ofantigenic materials. The influenza type virus used to prepare thevaccine composition will, of course, depend on the influenza againstwhich a recipient of the vaccine is to be protected.

For example, the non-live influenza virus antigen comprises one or moreantigens of, for instance, the genetic backbone of one or more of thefollowing influenza viruses: A/Ann Arbor/6/60 (A/AA/6/60) B/AnnArbor/1/66 virus, the FluMist MDV-A (ca A/Ann Arbor/6/60), the FluMistMDV-B (ca B/Ann Arbor/1/66), A/Leningrad/17 donor strain backbone, andPR8.

In another specific examples, the vaccine compositions of the inventioncomprise a non-live influenza virus antigen of, for instance, an HA oran NA polypeptide sequence (or at least 90% identical or at least 95%identical to such sequences) from one or more of the following:B/Yamanashi; A/New Caledonia; A/Sydney; A/Panama; B/Johannesburg;B/Victoria; B/Hong Kong; A/Shandong/9/93; A/Johannesburg/33/94;A/Wuhan/395/95; A/Sydney/05/97; A/Panama/2007/99; A/Wyoming/03/2003;A/Texas/36/91; A/Shenzhen/227/95; A/Beijing/262/95; A/NewCaledonia/20/99; B/Ann Arbor/1/94; B/Yamanashi/166/98;B_Johannesburg.sub.--5.sub.--99; BVictoria/504/2000; B/Hong Kong/330/01;B_Brisbane.sub.--32.sub.--2002; B/Jilin/20/03; an H1N1 influenza Avirus, an H3N2 influenza A virus, H9N2 influenza A virus, an H5N1influenza A virus; an H7N9 influenza A virus; an influenza B virus; anda pandemic influenza strain (either designated by WHO or not circulatingin the human population).

In one embodiment the influenza virus strain may be of one or more ofthe strains previously recommended by the WHO for use in an influenzavaccine.

The adjuvant of the composition of the invention is critical for itssuitability for intranasal administration and for its efficacy. Asuitable adjuvant for intranasal administration may be an adjuvant thatcomprises optionally a monoester of glycerol in combination with a fattyacid, or it may be a combination of fatty acids. Carboxylic acids usedin such adjuvants comprise long chain (C4-C30) alkyl, alkenyl or alkynylcarboxylic acids which may optionally be branched or unbranched, cyclicor acyclic, optionally having single, double or multiple unsaturation(double or triple bond) which may further optionally be of differentkind.

Monoglycerides used in such adjuvants may be carboxylic acid esters ofglycerin, wherein the carboxylic acids may be long chain (C4-C30) alkyl,alkenyl or alkynyl carboxylic acids which may optionally be branched orunbranched, optionally having single, double or multiple unsaturation(double or triple bond) which may further optionally be of differentkind.

The concentration of monoglyceride in a vaccine composition may be inthe range of e.g. about 1 to about 50 mg/ml, such as, e.g. from about 1to about 25 mg/ml, from about 5 to about 15 mg/ml or about 10 mg/ml.

The concentration of fatty acid in a vaccine composition may be in therange of e.g. about 0.5 to about 50 mg/ml, such as, e.g. from about 1 toabout 25 mg/ml, from about 1 to about 15 mg/ml, from about 1 to about 10mg/ml, from about 2 to about 8 mg/ml or about 6-7 mg/ml. In oneembodiment on a molar basis the concentration of a fatty acid in thevaccine composition corresponds to the concentration (on a molar basis)of the monoglyceride.

Any combination of the concentration ranges mentioned above formonoglyceride and fatty acid is within the context of the presentapplication. Moreover, the broadest range mentioned gives a preferredrange, and then the range is narrowed to the most preferred range.

The inventors of the present invention have found that adjuvants asdescribed above and disclosed in WO 2012/042003 (which is herebyincluded in its entirety by reference) are particularly useful whenvaccination is performed via the nasal route, e.g. administration to themucosa of the nasal cavity. The inventors have found that use of suchadjuvants in vaccination via the nasal route improves the immuneresponse upon vaccination. The inventors have found the use of suchadjuvants safe and tolerable in several species including humans.

Accordingly, the composition may comprise mono-glycerides which areglycerides mono-esterified with carboxylic acids selected from the groupconsisting of lauric acid (C12), myristic acid (C14), palmitic acid(C16), palmitoleic acid (016:1), oleic acid (018:1), linoleic acid(018:2), stearic acid, hexanoic acid, caprylic acid, decanoic acid(capric acid), arachidic acid, behenic acid, lignoceric acid,alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid,docosahexaenoic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid,arachidonic acid, erucic acid, nervonic acid.

In a further embodiment, the mono-glycerides are glyceridesmono-esterified with carboxylic acids selected from the group consistingof palmitoleic acid (016:1), oleic acid (C18:1) and linoleic acid(018:2).

Preferably, the mono-glyceride is glyceride mono-esterified with oleicacid (glyceryl oleate).

The adjuvant preferably comprises one or more carboxylic acids selectedfrom the group consisting of lauric acid, myristic acid, palmitic acid,palmitoleic acid, oleic acid, linoleic acid stearic acid, hexanoic acid,caprylic acid, decanoic acid (capric acid), arachidic acid, behenicacid, lignoceric acid, alpha-linolenic acid, stearidonic acid,eicosapentaenoic acid, docosahexaenoic acid, gamma-linolenic acid,dihomo-gamma-linolenic acid, arachidonic acid, erucic acid and nervonicacid. Preferably, the one or more carboxylic acids are selected from thegroup consisting of oleic acid and lauric acid.

In a combination of suitable embodiments, the adjuvant comprisesglyceryl oleate, oleic acid and an aqeuous medium. The vaccinecomposition of the present invention can also comprise additionalpharmaceutical excipients. Such pharmaceutical excipients can be:

1. Agents to control the tonicity/osmolarity of the vaccine. Such agentsare e.g. physiological salts like sodium chloride. Other physiologicalsalts are potassium chloride, potassium dihydrogen phosphate, disodiumphosphate, magnesium chloride etc. Such agent could also be other ionicsubstances which influence the ionic strength and stability. Theosmolality of the vaccine may be adjusted to a value in a range fromabout 200 to about 400 mOsm/kg, preferably in a range from about 240 toabout 360 mOsm/kg or the osmolality must be close to the physiologicallevel e.g. in the physiological range from about 290 to about 310mOsm/kg.

2. Agents to adjust the pH of or to buffer the vaccine composition.Normally, pH of the vaccine composition is in a range of from about 5 toabout 8.5. Suitable pH adjusting agents or buffer substances includehydrochloric acid, sodium hydroxide (to adjust pH) as well as phosphatebuffer, Tris buffer, citrate buffer, acetate buffer, histidine bufferetc. (to buffer the vaccine).

3. Other additives like e.g. surface-active agents, antioxidants,chelating agents, antibacterial agents, viral inactivators,preservatives, dyes, anti-foaming agents, stabilizers or surface activeagents, or combinations thereof.

The surface-active agent may be hydrophilic, inert and biocompatible,such as, e.g., poloxamers such as e.g. Pluronic F68 or Pluronic 127.

The antibacterial agents may be e.g. amphotericin or any derivativethereof, chlorotetracyclin, formaldehyde or formalin, gentamicin,neomycin, polymyxin B or any derivative thereof, streptomycin or anycombination thereof.

The antioxidants may be e.g. ascorbic acid or tocopherol or anycombination thereof.

The viral inactivators may be e.g. formalin, beta-propiolactone,UV-radiation, heating or any combination thereof.

The preservatives may be e.g. benzethonium chloride, EDTA, phenol,2-phenoxyethanol or thimerosal or any combination thereof. EDTA has alsobeen shown to be a chelating agent, an antioxidant and a stabilizer.

The dyes may be e.g. any indicators (such as e.g. phenol red) orbrilliant green or any combination thereof.

The anti-foaming agents may be e.g. polydimethylsilozone.

The surfactants may be e.g. anionic, cationic or non-ionic orzwitterionic, such as e.g. polyoxyethylene and derivatives thereof,polysorbates (such as e.g. polysorbate 20 or polysorbate 80), Tween 80,poloxamers (such as e.g Pluronic F68) or any combination thereof.

Typically, the concentration of monoglyceride in a vaccine compositionis in an amount in the range of about 0.1 g to about 5.0 g per 100 mL,or in the range of about 0.1 g about 2.0 g per 100 ml, or about 0.5 g toabout 2.0 g, such as 0.5 g to about 1.5 g per 100 mL of the vaccinecomposition.

Furthermore, the concentration of the one or more carboxylic acids is inan amount in the range of about from 0.1 g to about 5.0 g per 100 mL, orin the range of about 0.1 g to about 2.0 g per 100 mL or about 0.5 g toabout 2.0 g, such as 0.5 g to about 1.5 g per 100 mL of the vaccinecomposition.

The one or more monoglycerides together with one or more carboxylicacids in an vaccine composition may be in an amount of at the most 10%w/v, or at the most 5% w/v, or at the most 4% w/v, or at the most 3%w/v, or at the most 2% w/v, or at the most 1% w/v, or at the most 0.5%w/v, or at the most 0.1% w/v, or at the most 0.05% w/v.

The adjuvant may comprise a combination of lipids selected from thegroup consisting of mono-olein, oleic acid, lauric acid, and soybeanoil. In one suitable embodiment, the adjuvant comprises oleic acid,lauric acid in Tris buffer. Suitably, this embodiment comprises 0.25 gto 0.75 g of oleic acid, 0.25 g to 0.75 g of lauric acid in 7-15 mL ofTris buffer (pH 7-9). A specific example comprises 0.4 g to 0.5 g ofoleic acid, 0.3 g to 0.4 g of lauric acid in 8-10 mL of 0.1 MTris buffer(pH 7-9). In a further suitable embodiment, the adjuvant comprises oleicacid and mono-olein in Tris buffer. Suitably, this embodiment comprises0.25 g to 0.75 g of oleic acid, 0.25 g to 0.75 g of mono-olein in 7-15mL of Tris buffer. A specific example comprises 0.3 g to 0.4 g of oleicacid, 04 g to 0.5 g of mono-olein in 8-10 mL of 0.1 MTris buffer (pH7-9). A further embodiment comprises 0.5 g to 0.25 g of mono-olein, 0.5g to 0.25 g of oleic acid, and 0.25 g to 0.75 g of soybean oil in 7-15mL of Tris buffer. A specific example of this embodiment comprises 0.1 gto 0.2 g of mono-olein, 0.8 g to 1.5 g of oleic acid, and 0.5 g to 0.6 gof soybean oil in 8-12 mL of Tris buffer (pH 7-9).

Three types of adjuvants were used successfully in the examples below:Example adjuvant A comprising 0.4 g to 0.5 g of oleic acid, 0.3 g to 0.4g of lauric acid in 8-10 mL of 0.1 MTris buffer (pH 7-9); Exampleadjuvant B comprising 0.3 g to 0.4 g of oleic acid, 0.4 g to 0.5 g ofmono-olein in 8-10 mL of 0.1 MTris buffer (pH 7-9); and Example adjuvantC comprising 0.1 g to 0.2 g of mono-olein, 0.8 g to 1.5 g of oleic acid,and 0.5 g to 0.6 g of soybean oil in 8-12 mL of Tris buffer (pH 7-9).These adjuvants are typically prepared in w/v concentration of 2-12%lipid content (6 g-12 g per 100 mL), most typically from 3-10%, such as4%, 5%, 6%, 7, 8%, or 9%, These concentrations are those of the adjuvantmix itself. This adjuvant is then mixed with the antigen containingcomposition in 2:1 to 1:8 ratios, such as, for example, in a 1:1 ratioso as to provide a 4% lipid content vaccine composition when commencingfrom an adjuvant with an 8% lipid concentration. Typically, the lipidcontent in the vaccine composition of the invention is 0.5% to 6% w/v,typically as 1% to 6% w/v, more typically 1% to 4%.

The Example B composition is an Endocine™ formulation comprisingequimolar amounts of glycerol monooleate and oleic acid (0.3 g to 0.4 gof oleic acid, 0.4 g to 0.5 g of mono-olein in 8-10 mL of 0.1 M Trisbuffer (pH 7-9)) and has been found to be exceptionally effective innaive subjects with no pre-existing immunity to the antigen. In a highlypreferred embodiment, this 8% lipid formulation is diluted with theantigen containing compositions so as to provide a vaccine compositionwith a lipid concentration of 1-4% w/v.

As stated, the composition is suitable for use in a method forimmunization during a peri-pandemic or pandemic period comprisingintranasally administering the vaccine composition of the invention. Themethod for immunization during a pen-pandemic or pandemic period can beused for subjects of all age. The invention further relates to a methodof immunization during seasonal epidemics of immuno-compromised subjectscomprising intranasally administering a vaccine composition asdescribed.

As stated, the invention is directed to a method of immunizationimmuno-compromised subjects comprising intranasally administering avaccine composition.

The Examples below show the efficacy of this vaccine composition ininfluenza naive subjects (ferrets) and immuno-compromised such as theelderly.

The surprisingly efficacy in eliciting an immune response in naïveindividuals implies that the vaccine of the invention is able to elicitimmune response in individuals who have a weakened immune system interms of being able to respond to invasive vira where do they do notalready have strong immunoprotection. Immuno-compromised individualswill greatly benefit from the vaccine composition of the invention.Accordingly, a further aspect of the invention is directed to adjuvantednon-live antigens against influenza intranasally administered toimmune-compromised patients, including those with immunosenescence; HIVpatients; subjects taking immunosuppressant drugs, recent organrecipients; premature babies, and post-operative patients. This aspectrelates to a composition comprising

-   -   i) one or more non-live antigens, and    -   ii) an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides        for use as an intranasally administered vaccine, wherein said        vaccine is for immunization of immuno-compromised subjects.

A surprising effect of the present invention as illustrated by example 2is that the composition of the present invention is able to reduce virusshedding. Immuno-compromised subjects shed more virus thanimmune-competent healthy adults. Immuno-compromised subjects aretherefore able to spread more virus to people in their proximity such ascare takers, family, residents at nursing homes. The present inventionmay therefore be suitable for treating immuno-compromised subjects suchas individuals aged ≧65 years, pregnant women, cancer patients, patientsreceiving chemotherapy, radiation therapy, HIV infected individuals. Thepresent invention may be suitable for preventing virus spreading byimmuno-compromised subjects as identified in table 1. In one embodimentthe composition of the present invention is for use inimmuno-compromised individuals aged ≧65 years for reducing virusshedding. In one embodiment the composition of the present invention isfor use in pregnant women for reducing virus shedding. In one embodimentthe composition of the present invention is for use in HIV infectedsubjects for reducing virus shedding. In one embodiment the compositionof the present invention in for use in persons receivingimmunosuppressive medication e.g. glucocorticoid therapy for reducingvirus shedding. Further, a composition of the percent invention may beparticularly suitable for containing a pandemic by reducing virusspreading. In one embodiment a composition of the present invention isfor use in immuno-compromised subjects for reducing virus shedding in apandemic zone. In one embodiment a composition of the present inventionis for use in immuno-compromised subjects for reducing virus sheddingduring a peri-pandemic period. In one embodiment a composition of thepresent invention is for use in the immuno-compromised subjects forreducing virus shedding during a peri-pandemic period.

A method of immunization against influenza in immuno-compromisedpatients by intranasal administration of a composition as describedsupra is an interesting aspect of the surprising result.

Approximately 90% of the more than 30.000 influenza related deaths peryear in the USA occur among persons of 65 years or older, illustratingthe high vulnerability of this population. Current influenza vaccineshave reduced effect in elderly (17-53%) compared to young adults(70-90%). The increased susceptibility to virus infection and thereduced efficacy of vaccination among the elderly population is due toimmunosenescence. Immunosenescence is a progressive age-dependentdecline in the function of the immune system affecting both innate andadaptive immunity. An essential part of the innate immune system is thepattern recognition receptors (PRR), which recognize conservedstructures of a broad array of pathogens. A class of PRRs know astoll-like receptors (TLR) are involved in recognizing influenza virus.Dendritic cells (DCs) form an essential bridge between the innate andadaptive immune system by expressing TLRs and capturing antigen. Severalstudies have demonstrated age-related changes in DC function e.g.reduced antigen capture capacity, reduced TLR-expression and function,impaired migration capacity and reduced T cell activating capacity.

Aging is also accompanied by a gradual decrease in naive B cells and anincrease in effector B cells, leading to reduced diversity and loweraffinity of the antibody response. The T cell compartment whereof thetwo major subsets are the CD4 and CD8 T cells are also greatly affectedby aging. The most dramatic change being the involution of the thymus,which results in a reduction of naive T cells in the periphery inelderly individuals. The reduced thymic output has a profound effect onthe T cell population resulting in decreased diversity in the T cellreceptor (TCR) repertoire. A reduction in the TCR repertoire has beenassociated with poor vaccination response and impaired immunity againstinfluenza virus. The age-related decline of the immune system's abilityto elicit an efficient response to pathogens is a complex phenomenonthat is caused by multiple changes in various cell types.

For these reasons, the elderly are a particularly vulnerable populationand there is a need for more efficient vaccines for this patient classas the present invention, illustrated by example 4.

As stated, an interesting aspect of the invention is directed to acomposition comprising one or more non-live influenza virus antigens,and

-   -   an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides        for use as an intranasally administered vaccine to        immuno-compromised subjects.

The composition is typically for use as an intranasally administeredvaccine to immuno-compromised subjects against infectious pathogens suchas influenza. The immune-compromised subjects are suitably selected fromthe group consisting of people but are not limited to subjects aged ≧65years, pregnant women, premature babies and following patient classes;cancer patients, persons receiving chemotherapy, persons receivingradiation therapy, organ transplant patients, persons undergoing solidorgan transplants, stem cell transplant patients, persons undergoinghematopietic allogenic stem cell transplantation, persons undergoinghematopoietic autologous stem cell transplantation. HIV infectedpatients, persons with AIDS, patients with graft-versus-host disease,patients on immune suppressive drugs e.g. glucocorticoid therapy andsteroid therapy.

As stated, immunosenescence is commonly found in the elderly.Accordingly, one interesting embodiment of the invention relates to acomposition for use as an intranasally administered vaccine in elderlysubjects, such as aged 55 or more, typically aged 60 or more, mosttypically aged 65 or more, such as aged 75 or more, such as aged 80 ormore, such as aged 85 or more, such as aged 90 or more, said compositionas described herein.

A further aspect of the invention is directed to a vaccine for use innaive subjects such as pediatric subjects who are alsoimmuno-compromised patients. The adjuvant of the invention hasdemonstrated its efficacy in naive subjects in influenza. This rendersit suitable for both naive patient classes and immuno-compromisedpatients in general.

Accordingly, another aspect of the invention is directed to acomposition for use as an intranasally administered vaccine for use inpediatric immuno-compromised patients, said composition comprising

-   -   i) one or more non-live antigens, and    -   ii) an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides.

Suitable types of vaccines for immunization of naive subjects andpediatric immuno-compromised patients comprise, according to the presentinvention, an antigen of the respectively relevant pathogen intended tobe immunized or treated by vaccine. This includes, without being limitedto, immunogens derived from viruses selected from the group consistingof hepatitis B, hepatitis A, hepatitis C, hepatitis D & E virus,Non-A/Non-B Hepatitis virus, pox and smallpox viruses, polio virus,measles virus, human immunodeficiency virus (HIV), enteroviruses,retroviruses, respiratory syncytial virus, rotavirus, human papillomavirus, varicella-zoster virus, yellow fever virus, SARS virus, animalviruses, herpes viruses, cytomegalovirus, varicella zoster, Epstein Barrvirus, para-influenza viruses, adenoviruses, coxsakie viruses, picornaviruses, rhinoviruses, rubella virus, papovirus, and mumps virus. Somenon-limiting examples of known viral antigens other than the Influenzavirus antigens mentioned above may include the following: antigensderived from HIV-I such as tat, nef, gpl20 or gpl[beta]O, gp40, p24,gag, env, vif, vpr, vpu, rev or part and/or combinations thereof;antigens derived from human herpes viruses such as gH, gL gM gB gC gK gEor gD or or part and/or combinations thereof or Immediate Early proteinsuch as ICP27, ICP47, ICP4, ICP36 from HSVI or HSV2; antigens derivedfrom cytomegalovirus, especially human cytomegalovirus such as gB orderivatives thereof; antigens derived from Epstein Barr virus such asgp350 or derivatives thereof; antigens derived from Varicella ZosterVirus such as gp I, 11, 111 and IE63; antigens derived from a hepatitisvirus such as hepatitis B, hepatitis C or hepatitis E virus antigen(e.g. env protein EI or E2, core protein, NS2, NS3, NS4a, NS4b, NS5a,NS5b, p7, or part and/or combinations thereof of HCV); antigens derivedfrom human papilloma viruses (for example HPV6, 11, 16, 18, e.g. LI, L2,EI, E2, E3, E4, E5, E6, E7, or part and/or combinations thereof);antigens derived from other viral pathogens, such as RespiratorySyncytial virus (e.g F and G proteins or derivatives thereof),parainfluenza virus, measles virus, mumps virus, flaviviruses (e. g.Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus) or part and/or combinations thereof.

The antigens may be e.g. whole non-live antigens such as e.g. wholeinactivated viruses. The antigen may also be part of a pathogen such ase.g. part of an inactivated virus. The antigen components that may beused are, but not limited to, for example, viral, bacterial,mycobaterial or parasitic antigens. Bacterial pathogens may be e.g.Mycobacteria causing tuberculosis and leprosy, pneumocci, aerobic gramnegative or gram-positive bacilli, mycoplasma, staphyloccocalinfections, streptococcal infections, Helicobacter pylori, salmonellaeand chlamydiae. The diseases may also be bacterial infections such asinfections caused by Mycobacteria causing tuberculosis and leprosy,pneumocci, aerobic gram negative bacilli, mycoplasma, staphyloccocalinfections, streptococcal infections, Helicobacter pylori, salmonellae,diphtheria and chlamydiae.

Preferred types of vaccines for immunization of immuno-compromisedpatients may be selected from the group consisting of pneumococcalvaccine, Hepatitis A-E vaccine, Meningococci vaccine, Haemophilusinfluenzae b (Hib) vaccine, Diphtheria vaccine.

The diseases may also be parasitic malaria, leishmaniasis,trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis or varioustypes of cancer such as, e.g. breast cancer, stomach cancer, coloncancer, rectal cancer, cancer of the head and neck, renal cancer,malignant melanoma, laryngeal cancer, ovarian cancer, cervical cancer,prostate cancer.

The diseases may also be allergies due to house dust mite, pollen andother environmental allergens and autoimmune diseases such as, e.g.systemic lupus erythematosis.

The antigen in the vaccine composition may be whole non-live antigenssuch as e.g. whole inactivated viruses, split non-live antigens orsubunit non-live antigens. Inactivation processes are well known in theart such as heat inactivation, irradiation inactivation by UV-light orin activation by formalin inactivation or treatment withbeta-propiolactone.

The composition of the invention are for use as vaccines forimmunization of immuno-compromised patients. The immuno-compromisedpatients are suitably selected from the group consisting of people withimmunosenescence; HIV infected subjects; subjects takingimmunosuppressant drugs, such as recent organ recipients; prematurebabies, and post-operative patients. As stated, immunosenescence iscommonly found in the elderly. Accordingly, one interesting embodimentof the invention relates to a composition for use as an intranasallyadministered vaccine in elderly subjects, such as aged 55 or more,typically aged 60 or more, most typically aged 65 or more, saidcomposition as described herein. The immuno-compromised pediatricsubjects may be children under 18 years old, such as children 0 to 18years, particularly children aged 12 and under. The inventionparticularly intended for immuno-compromised children less than 8 yearsof age, such as 6 years old or less. An important intended class ofpatients for the vaccine of the invention is particularlyimmuno-compromised children of 2 months to less than 9 years of age,typically children of age 3 months to less than 9 years old, such as ofage 6 months to less than 8 years old, most typically of age 6 month toless than 7 years old, such as of age 6 months to less than 72 months,or of age 6 months to 60 months or of age 6 months to 24 months. Thecomposition of the invention is intended, at least in part, as a vaccinefor pediatric use in immune-compromised subjects.

The immuno-compromised subjects may be of all age groups when thecomposition is particularly directed to a vaccine for use duringpandemic or peri-pandemic period.

Streptococcus pneumoniae is a major cause of morbidity and mortalityworldwide with an estimated 1.6 million people dying of invasivepneumococcal disease (IPD) each year (WHO, 2002). IPD occurs mostcommonly among the very young (<24 months) and the elderly (>65 years);the elderly have the highest IPD mortality rates. Currently, fourvaccines are available for the prevention of infection withStreptococcus pneumoniae. No intranasal vaccines are available forStreptococcus pneumonia.

One interesting embodiment of the invention is directed to an intranasalalternative for the prevention of infection with Streptococcuspneumoniae, directed particularly at children and other naive subjectsand the elderly since this later group is known to beimmuno-compromised. The composition of the invention does not utilizelive attenuated bacteria but rather non-live streptococcus pneumoniaantigens. The surprisingly efficacy of the vaccine of the invention is aresult of the adjuvant used and the surprising result was specific fornaive subjects. Similar results are anticipated also forimmuno-compromised subjects.

Accordingly, a further aspect of the invention is directed to acomposition comprising

-   -   i) one or more non-live Streptococcus pneumoniae antigens, and    -   ii) an adjuvant comprising:    -   one or more carboxylic acids,    -   an aqueous medium, and    -   optionally one or more mono-glycerides        for use as an intranasally administered vaccine for use in        immuno-compromised subjectsfor the prevention of infection with        Streptococcus pneumoniae or for reducing the severity of        symptoms associated with an infection with Streptococcus        pneumoniae.

The immuno-compromised patients are suitably selected from the groupconsisting of people with immunosenescence; HIV infected subjects;subjects taking immunosuppressant drugs, such as recent organrecipients; premature babies, and post-operative patients. As stated,immunosenescence is commonly found in the elderly. Accordingly, oneinteresting embodiment of the invention relates to a composition for useas an intranasally administered vaccine in elderly subjects, such asaged 55 or more, typically aged 60 or more, most typically aged 65 ormore, said composition as described herein.

An important embodiment of the invention is directed to a vaccineagainst pneumococcal infection for the prevention of and/or reducing ofthe symptoms of disease states selected from the group consisting ofbronchitis, pneumonia, septicemia, pericarditis, meningitis andperitonitis.

One embodiment is related to the use of pneumococcal vaccine, such as apneumococcal polysaccharide vaccine (PPV) in immuno-compromisedsubjects, particularly for the elderly over the age of 60 or 65 yearsand/or adults with a history of previous pneumococcal infection oradults with an increased risk (e.g. anatomic or functional asplenia,immuno-compromising condition, or cardiac, liver, pulmonary, or renalchronic diseases, or recipients of organ, bone marrow, or cochleartransplants).

In a further embodiment, a pneumococcal vaccine composition of theinvention is used in subjects from 4 weeks of age to 6 years of age(e.g. to subjects that are naïve and with immune systems not fullydeveloped “immuno-compromised”) and to elderly, such as persons over 50years old, typically 60 years old or more, more typically 65 years oldor more.

The vaccine composition according to the invention may further comprisepharmaceutically acceptable excipients such as e.g. a medium which maybe an aqueous medium further comprising a surface-active agent, whichmay be hydrophilic and inert and biocompatible, such as, e.g.,poloxamers such as e.g. Pluronic F68 or Pluronic 127.

A pneumococcal vaccine according to present invention may furthercomprise antibacterial agents, antioxidants, viral inactivators,preservatives, dyes, stabilizers, anti-foaming agents, surfactants(non-ionic, anionic or cationic) as described herein, or any combinationthereof. The antibacterial agents may be e.g. amphotericin or anyderivative thereof, chlorotetracyclin, formaldehyde or formalin,gentamicin, neomycin, polymyxin B or any derivative thereof,streptomycin or any combination thereof. The antioxidants may be e.g.ascorbic acid or tocopherol or any combination thereof. The viralinactivators may be e.g. formalin, beta-propiolactone, UV-radiation,heating or any combination thereof.

When describing the embodiments of the present invention, thecombinations and permutations of all possible embodiments have not beenexplicitly described. Nevertheless, the mere fact that certain measuresare recited in mutually different dependent claims or described indifferent embodiments does not indicate that a combination of thesemeasures cannot be used to advantage. The present invention envisagesall possible combinations and permutations of the described embodiments.

EXAMPLES Example 1 Objective

The objective of the present study was to investigate the immunogenicityand protective efficacy of intranasally administered adjuvant-formulatedinfluenza split antigen and adjuvant-formulated killed whole influenzavirus antigen in the ferret model, according to the present invention.

The vaccine based on H1N1/California/2009 split antigen (vaccine A) wasstudied with antigen doses of 5, 15, or 30 μg HA and the vaccine basedon H1N1/California/2009 killed whole virus antigen (vaccine B) wasstudied with an antigen dose of 15 μg HA. Vaccine efficacy was studiedusing wild-type H1N1 A/The Netherlands/602/2009 virus as challenge.

The Endocine™ adjuvant comprised equimolar amounts of glycerolmonooleate and oleic acid with a final concentration of 20 mg/ml (2%) inthe vaccine composition. In this experiment Immunose™ FLU means non-liveinfluenza antigens mixed with Endocine™.

Experimental Groups Immunization Phase

TABLE 2 Antigen Group Number of Test dose (μg Route of number animalssubstance HA, H1N1) immunization 1 6 Saline 0 Nasal 2 6 Fluarix ® 15Subcutaneous 3 6 Vaccine A 5 Nasal 4 6 Vaccine A 15 Nasal 5 6 Vaccine A30 Nasal 6 6 Vaccine B 15 Nasal

Vaccine Preparation and Administration

Saline: 0.9% saline pH 5-5.5.

Fluarix®: Parenteral vaccine (composed ofA/California/7/2009(H1N1)-like, A/Perth/16/2009(H3N2)-like andB/Brisbane/60/2008-like vaccine strains at 15 μg HA of each vaccinestrain). Animals of group 2 were vaccinated subcutaneously at day 21 and42 with 0.5 ml Fluarix (GlaxoSmithKline Biologicals).

Vaccine A: Influenza vaccine nasal drops, 5, 15 and 30 μg HA/0.2 ml,adjuvant formulation comprisingan Endocine™ formulation of equimolaramounts of glycerol monooleate and oleic acid (pH 8, in Tris 0.1 M) witha final concentration of 20 mg/ml in the vaccine composition;H1N1/California/2009 split antigen.

Vaccine B: Influenza vaccine nasal drops, 15 μg HA/0.2 ml, adjuvantformulation comprising an Endocine™ formulation of equimolar amounts ofglycerol monooleate and oleic acid (pH 8, in Tris 0.1 M) with a finalconcentration of 20 mg/ml in the vaccine composition,H1N1/California/2009 killed whole virus antigen.

Ferrets

Healthy female ferrets (Mustela putorius furo: outbred), approximately12 months of age, with body weights of 760-1210 g and seronegative forantibodies against circulating influenza viruses B, A/H1N1, A/H3N2 andA/pH1N1 as demonstrated by hemagglutination inhibition (HI) assay wereused. Animals were housed in normal cages, in groups of maximal 8animals during the pre-immunization phase and in study groups of 6animals during the immunization phase. The study groups were transferredto negatively pressurized glovebox isolator cages on the day ofchallenge. During the whole study animals were provided with commercialfood pellets and water ad libitum.

Immunization

Five groups of six ferrets received three intranasal immunizations(droplets: 100 μl in each nostril, using a pipet with filtertip) underanesthesia with ketamine and domitor at days 0, 21 and 42. Animals ofgroup 1 received 200 μl of steril physiological saline (0.9% salinepH5-5.5). Groups 3, 4 and 5 were intranasally immunized with 200 μlEndocine™ formulated H1N1/California/2009 split antigen containing 5, 15and 30 μg HA, respectively. Group 6 was intranasally immunized with 200μl Endocine™ formulated H1N1/California/2009 whole virus antigencontaining 15 μg HA. Control group 1 received 200 μl of salineintranasally. One group of six ferrets (group 2) were vaccinatedsubcutaneously at day 21 and 42 with 0.5 ml Fluarix® (GlaxoSmithKlineBiologicals), season 2010/2011, a non-adjuvanted trivalent influenzavaccine (TIV) that contained 15 μg HA of each vaccine strain. Bloodsamples for serum preparation were collected prior immunization on days0, 21 and 42 and before challenge on study days 64 and 70.

Challenge Virus Preparation and Administration

On study day 70, all animals were challenged with a field isolate ofinfluenza virus (H1N1 strain A/The Netherlands/602/2009) by theintratracheal route. To prepare the challenge virus, the H1N1 A/TheNetherlands/602/2009 challenge stock (7.8 log 10 TCID50/ml) was dilutedin ice-cold PBS to a concentration of 3.3×105 TCID50/ml. All animalswere challenged intratracheally with 3 ml of the challenge viruspreparation containing 106 TCID50, administered with a small catheterinto the trachea using a tracheoscope and released just above thebifurcation. Preparation and administration of the challenge virus wereperformed under BSL3 conditions. One day after challenge a sample of theremaining challenge virus dilution was titrated on Madin-Darby caninekidney (MDCK) cells to confirm the infectivity of the virus. Backtitration of the challenge dilution one day after the inoculation showedthat the material still contained 4.8 log 10 TCID50.

Procedures and Sample Collection

Several procedures were performed on the ferrets over the course of theexperiment. For implantation of temperature sensors, immunizations,viral challenge and computed tomography (CT) imaging the animals wereanesthetized with a cocktail of ketamine (4-8 mg/kg: i.m.; Alfasan,Woerden, The Netherlands) and domitor (0.1 mg/kg: i.m.; Orion Pharma,Espoo, Finland). For sampling (blood, swabs and nasal washes) andeuthanasia by exsanguination, the animals were anesthetized withketamin. Two weeks prior to the start of the experiment, a temperaturelogger (DST micro-T ultrasmall temperature logger; Star-Oddi, Reykjavik,Iceland) was placed in the peritoneal cavity of the ferrets. This devicerecorded body temperature of the animals every 10 minutes. Ferrets wereweighed prior to each immunization (days 0, 21 and 42) and on the daysof challenge and euthanasia (days 70 and 74). Animals of groups 1, 2 and4 were monitored by CT imaging on days 64, 71, 72, 73 and 74. Bloodsamples were collected prior to the immunization on days 0, 21 and 42,on day 64 and before challenge on day 70. Nose and throat swabs werecollected prior challenge on day 70 and on each day after challenge.

Collection of Blood Samples and Serum

Blood samples were collected and split in 2 equal volumes. One volume,used to isolate PBMC, was immediately transferred to a tube containingEDTA anti-coagulant. The other volume, used to collect serum, wastransferred to a serum tube containing clot activator. All serum tubeswere centrifuged at ca. 2000×g for 10 minutes at room temperature. Serumwas aliquoted in 0.1 ml samples and stored at ca. −80° C.

Isolation of PBMC and Plasma

Blood samples, used to isolate PBMC, were immediately transferred to atube containing EDTA anti-coagulant, centrifuged at 880×G for 5 min, theplasma was stored at ca. −80° C. The cell pellet was resuspended in 3.5ml wash buffer (D-PBS: lot#: RNBB7791, V-CMS: 10700395 and EDTA:lot#:079K8712, V-CMS: 10700037), layered on 3 ml lymphoprep and centrifugedat 800×G for 30 minutes. After centrifugation the cell containinginterface was collected, transferred to a new tube and 4 times washed inwash buffer. Centrifugation at 600×g, 465×g and 350×g for 10 min and at250×g for 15 min was involved in the subsequent washing steps. After thelast wash step, the cell pellet was resuspended, put on ice for at least10 min, resupended in 1 ml ice cold freeze medium (RPMI lot#1MB078, 20%FCS VC#201110194, 10% DMSO VC #10700203), transferred to an ampoule, andstored at −80° C.

Serology

Antibody titers against H1N1 A/The Netherlands/602/2009 and 2 distantviruses H1N1 A/Swine/Ned/25/80 and H1N1 A/Swine/Italy/14432/76 weredetermined by hemagglutination inhibition assay (HI) and virusneutralization assay (VN). Antibody titers against the distant virusH1N1 A/New Jersey/08/76 were determined by hemagglutination inhibitionassay.

HI Assay

The HI assay is a standard binding assay based on the ability ofinfluenza virus hemagglutinin specific antibodies to block influenzainduced agglutination of red blood cells. The samples were pre-treatedwith cholera filtrate (obtained from Vibrio cholerae cultures) in orderto remove non-specific anti-hemagglutinin activity. Following anincubation for 16 hours at 37° C. the cholera filtrate was inactivatedby incubating the samples for 1 hour at 56° C. Serial two-fold dilutionsof the samples were made in phosphate buffered sulphate (PBS) (induplicate 96-wells plates starting with a dilution of 1:20) and when thesamples showed a-specific hemagglutination, they were pre-treated withturkey erythrocytes. After removal of these erythrocytes the sampleswere incubated with a fixed concentration of 4 hemagglutination units(HAU) of the concerning influenza virus for 1 hour at 4° C. Finally, theplates were scored independently by two technicians for inhibition ofhemagglutination, as shown by sedimentation of the erythrocytes.Trending ferret control sera were included in all runs.

VN Assay

The VN assay is a standard assay based on the ability of a subset ofinfluenza virus-specific antibodies to neutralize the virus such thatthere will be no virus replication in the cell culture. The samples wereheat-inactivated for 30 minutes at 56° C. and subsequently serialtwo-fold dilutions of the samples were made in infection medium (Eaglesminimal essential medium supplemented with 20 mM Hepes, 0.075% sodiumbicarbonate, 2 mM L-Glutamine, 100 IU/ml of penicillin and streptomycin,17.5 μg/ml trypsin and 2.3 ng/ml amphotericin B) in triplicate in96-wells plates starting with a dilution of 1:8. The sample dilutionswere then incubated with 25-400 TCID50 of the concerning virus for 1hour at 37° C., 5% CO2. After completion of the 1 hour incubation periodthe virus-antibody mixtures were transferred to plates with Madine DarbyCanine Kidney (MDCK) cell culture monolayers that were 95-100%confluent. These plates were than incubated for 1 hour at 37° C., 5%CO2, and the virus-antibody mixtures were subsequently removed andreplaced by infection medium. After an incubation period of 6 days at37° C., 5% CO2 the plates were read using turkey erythrocytes to detectthe presence of influenza virus hemagglutinin. The VN titers werecalculated according to the method described by Reed and Muench (Reed,L. J.; Muench, H. (1938). “A simple method of estimating fifty percentendpoints”. The American Journal of Hygiene 27: 493-497).

Virus Replication in the Upper and Lower Respiratory Tract

On days 0, 1, 2, 3 and 4 after challenge, nose and throat swabs weretaken from the animals under anesthesia. Four days after challenge, theferrets were euthanized by exsanguination under anesthesia after whichfull-body gross-pathology was performed and tissues were collected.Samples of the right nose turbinate and of all lobes of the right lungand the accessory lobe were collected and stored at −80° C. untilfurther processing. Turbinate and lung samples were weighed andsubsequently homogenized with a FastPrep-24 (MP Biomedicals, Eindhoven,The Netherlands) in Hank's balanced salt solution containing 0.5%lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 μg/ml streptomycin,100 U/ml polymyxin B sulfate, 250 μg/ml gentamycin, and 50 U/ml nystatin(ICN Pharmaceuticals, Zoetermeer, The Netherlands) and centrifugedbriefly before dilution.

After collection, nose and throat swabs were stored at −80° C. in thesame medium as used for the processing of the tissue samples.Quadruplicate 10-fold serial dilutions of lung and swab supernatantswere used to determine the virus titers in confluent layers of MDCKcells as described previously (Rimmelzwaan G F et al., J Virol Methods1998 September; 74(1)57-66).

Antibody Titer Results

Serum levels of antibodies were determined on days 0, 21, 42, 64, and 70prior to each immunization. Titers against H1N1 A/TheNetherlands/602/2009 and 2 distant viruses (H1N1 A/Swine/Ned/25/80 andH1N1 A/Swine/Italy/14432/76 were determined by hemagglutinationinhibition assay (HI) and virus neutralization assay (VNT). Antibodytiters against the distant virus H1N1 A/New Jersey/08/76) weredetermined by hemagglutination inhibition assay (HI).

HI antibody titers—Homologous: H1N1 A/The Netherlands/602/2009

The geometric mean HI titers are depicted in FIG. 1. The ≦5 value wasreplaced with the corresponding absolute value 5 for calculation of thegeometric mean. All pre-sera (day 0) were HI antibody negative (titer:≦5).

Analysis of the HI titers by group revealed the following results:

Group 1 (Saline; infection control)

All serum samples were HI antibody negative.

Group 2 (Fluarix®; parenteral control)

One serum sample collected after the first immunization (day 42) was lowHI antibody positive (titer: 13). Low HI titers (range 13-70) weredetected after the second immunization in sera of five out of sixanimals.

Group 3 (Vaccine A, 5 μg HA; intranasal)

All samples collected after the first immunization were HI antibodypositive (day 21; GMT: 477, range 160-1120). HI antibody titersincreased considerably after the second immunization (day 42; GMT: 1669,range 1120-2560) and in four out of six animals also after the thirdimmunization (day 64; GMT: 2158, range 1280-3840). Samples collected onday 70 (day of challenge) showed HI titers comparable to those measuredat day 64 (day 70; GMT: 2103, range 1120-3840).

Group 4 (Vaccine A, 15 μg HA; intranasal)

Five out of six samples collected after the first immunization were HIantibody positive (day 21; GMT: 1130 range, 5-5760). All samplescollected after the second immunization were HI antibody positive; HIantibody titers increased considerably in five animals (day 42; GMT:3673, range, 1120-5760). The third immunization did not result inincreased HI antibody titers (day 64; GMT: 2386, range 1920-4480).Samples collected on day 70 (day of challenge) showed HI titerscomparable to those measured at day 64 (day 70; GMT: 2281, range1280-2560).

Group 5 (Vaccine A, 30 μg HA; intranasal)

All samples collected after the first immunization were HI antibodypositive (day 21; GMT: 1249, range 400-3200). HI antibody titersincreased in five out of six animals after the second immunization (day42; GMT: 1874, range 640-3840) and in two animals also after the thirdimmunization (day 64; GMT: 1837 range 1280-3200). Samples collected onday 70 (day of challenge) showed HI titers comparable to those measuredat day 64 (day 70; GMT: 1699, range 640-3200).

Group 6 (Vaccine B, 15 μg HA; intranasal)

Five out of six samples collected after the first immunization were HIantibody positive (day 21; GMT: 87, range 5-1280). HI antibody titersincreased considerably in all animals after the second immunization (day42; GMT: 577, range 100-2880) and in two animals also after the thirdimmunization (day 64; GMT: 626, range 160-2560). Samples collected onday 70 (day of challenge) showed HI titers comparable to those measuredat day 64 (day 70; GMT: 583, range 160-2240).

Heterologous: H1N1 A/Swine/Ned/25/80, H1N1 A/Swine/Italy/14432/76 andH1N1 A/New Jersey/08/76

HI antibody titers against the distant viruses H1N1 A/Swine/Ned/25/80,H1N1 A/Swine/Italy/14432/76 and H1N1 A/New Jersey/08/76 were detected.The geometric mean HI titers against the distant viruses are depicted inFIG. 2. The ≦5 value was replaced with the corresponding absolute value5 for calculation of the geometric mean. All pre-sera (day 0) were HIantibody negative (titer: ≦5). Cross-reactive HI antibody titers wereconsiderably lower than homologous H1N1 A/The Netherlands/602/2009 HIantibody titers.

Analysis of the HI titers by group revealed the following results:

Group 1 (Saline; infection control)

All serum samples were HI antibody negative, except one. One samplecollected on day 64 showed a very low HI antibody titer of 7.5 againstH1N1 A/Swine/Italy/14432/76.

Group 2 (Fluarix®; parenteral control)

All samples were H1N1 A/Swine/Ned/25/80 and H1N1 A/Swine/Italy/14432/76HI antibody negative. Low HI titers against H1N1 A/New Jersey/08/76 weredetected in three out of six animals after the first immunization insera collected on days 42.

Group 3 (Vaccine A, 5 μg HA; intranasal)

All animals developed cross-reactive HI antibodies against the threedistant viruses. The highest titers were measured after the secondand/or third immunization. H1N1 A/Swine/Ned/25/80 HI antibody titers(GMT) on days 21, 42, 64 and 70 were 6 (range 5-7.5), 24 (range 5-60),32 (range 20-80) and 19 (range 5-70), respectively. H1N1A/Swine/Italy/14432/76 HI antibody titers (GMT) on days 21, 42, 64 and70 were 16 (range 5-50), 38 (range 10-80), 63 (range 40-160) and 42(range 20-120), respectively. H1N1 A/New Jersey/08/76 HI antibody titers(GMT) on days 21, 42, 64 and 70 were 5, 26 (range 7.5-70), 39 (range5-80) and 29 (range 20-50), respectively.

Group 4 (Vaccine A, 15 μg HA; intranasal)

All animals developed cross-reactive HI antibodies against the threedistant viruses after the second immunization. The third immunizationdid not result in increased HI titers. H1N1 A/Swine/Ned/25/80 HIantibody titers (GMT) on days 21, 42, 64 and 70 were 42 (range 5-90),239 (range 20-1120), 88 (range 50-160) and 75 (range 40-160),respectively. H1N1 A/Swine/Italy/14432/76 HI antibody titers (GMT) ondays 21, 42, 64 and 70 were 78 (range 5-280), 327 (range 35-1280), 153(range 80-320) and 105 (range 70-160), respectively. H1N1 A/NewJersey/08/76 HI antibody titers (GMT) on days 21, 42, 64 and 70 were 25(range 5-80), 176 (range 60-400), 64 (range 40-140) and 63 (range40-160), respectively.

Group 5 (Vaccine A, 30 μg HA; intranasal)

All animals except one developed cross-reactive HI antibodies againstH1N1 A/Swine/Ned/25/80. All animals developed cross-reactive HIantibodies against H1N1 A/Swine/Italy/14432/76 and H1N1 A/NewJersey/08/76. The highest titers were measured after the second and/orthird immunization. H1N1 A/Swine/Ned/25/80 HI antibody titers (GMT) ondays 21, 42, 64 and 70 were 23 (range 5-80), 41 (range 5-320), 42 (range5-320) and 34 (range 5-320), respectively. H1N1 A/Swine/Italy/14432/76HI antibody titers (GMT) on days 21, 42, 64 and 70 were 39 (range5-160), 54 (range 5-640), 78 (range 20-720) 50 (range 5-480),respectively. H1N1 A/New Jersey/08/76 HI antibody titers (GMT) on days21, 42, 64 and 70 were 9 (range 5-30), 40 (range 5-400), 35 (range5-160) and 27 (range 5-160), respectively.

Group 6 (Vaccine B, 15 μg HA; intranasal)

All animals developed cross-reactive HI antibodies against H1N1A/Swine/Italy/14432/76. All animals except one developed cross-reactiveHI antibodies against H1N1 A/Swine/Ned/25/80 and all animals except onedeveloped cross-reactive HI antibodies against H1N1 A/New Jersey/08/76.The highest titers were measured after the second and/or thirdimmunization. H1N1 A/Swine/Ned/25/80 HI antibody titers (GMT) on days21, 42, 64 and 70 were 7 (range 5-40), 19 (range 5-80), 15 (range 5-80)and 9 (range 5-40), respectively. H1N1 A/Swine/Italy/14432/76 HIantibody titers (GMT) on days 21, 42, 64 and 70 were 9 (range 5-160), 32(range 5-160), 27 (range 5-160), 15 (range 5-80), respectively. H1N1A/New Jersey/08/76 HI antibody titers (GMT) on days 21, 42, 64 and 70were 8 (range 5-80), 47 (range 10-240), 19 (range 5-140) and 13 (range5-80), respectively.

VN Antibody Titers:

Homologous: H1N1 A/The Netherlands/602/2009

VN antibody titers were measured in serum samples from all experimentalanimals. The geometric mean VN titers are depicted in FIG. 3. Allpre-sera (day 0) were VN antibody negative (titer: ≦8).

Analysis of the VN titers by group revealed the following results:

Group 1 (Saline; infection control)

All serum samples were VN antibody negative, except one collected on day42 that measured ≦64.

Group 2 (Fluarix®; parenteral control)

All serum samples were VN antibody negative.

Group 3 (Vaccine A, 5 μg HA; intranasal)

Four out of six samples collected after the first immunization were lowVN antibody positive (day 21; GMT: 19 range, 8-64). All samplescollected after the second immunization were VN antibody positive. VNantibody titers increased considerably in five animals after the secondimmunization (day 42; GMT: 242, range, 64-859) and after the thirdimmunization (day 64; GMT: 995, range 362-2436). Samples collected onday 70 (day of challenge) showed comparable, or lower VN titers thanthose measured at day 64 (day 70; GMT: 535, range 304-859).

Group 4 (Vaccine A, 15 μg HA; intranasal)

Five out of six samples collected after the first immunization were VNantibody positive (day 21; GMT: 147 range, 8-724). All samples collectedafter the second immunization were VN antibody positive. VN antibodytiters increased considerably in five animals after the secondimmunization (day 42; GMT: 2376, range, 64-8192) and in two animalsafter the third immunization (day 64; GMT: 1688, range 662-4871).Samples collected on day 70 (day of challenge) showed VN titerscomparable to those measured at day 64 (day 70; GMT: 1581, range351-3444).

Group 5 (Vaccine A, 30 μg HA; intranasal)

All samples collected after the first immunization were VN antibodypositive (day 21; GMT: 74, range 11-627). VN antibody titers increasedconsiderably in five out of six animals after the second immunization(day 42; GMT: 504, range 41-3435) and in three out of six animals afterthe third immunization (day 64; GMT: 1673 range 724-4884). Samplescollected on day 70 (day of challenge) showed VN titers comparable tothose measured at day 64 (day 70; GMT: 1699, range 304-5793).

Group 6 (Vaccine B, 15 μg HA; intranasal)

Two out of six samples collected after the first immunization were lowVN antibody positive (day 21; GMT: 12, range 8-64). All samplescollected after the second immunization were VN antibody positive (day42; GMT: 78, range 32-304). VN antibody titers increased after the thirdimmunization (day 64; GMT: 242, range 113-747). Samples collected on day70 (day of challenge) showed comparable, or lower VN titers than thosemeasured at day 64 (day 70; GMT: 177, range 91-362). Heterologous: H1N1A/Swine/Ned/25/80, H1N1 A/Swine/Italy/14432/76. VN antibody titersagainst the distant viruses H1N1 A/Swine/Ned/25/80 and H1N1A/Swine/Italy/14432/76 were tested (data not shown). All groups 3, 4, 5,and 6 outperformed groups 1 and 2 on days 42, 64 and 70.

Example 2

For all experimental animals certain clinical and pathologicalparameters were determined, i.e. mortality, body temperature, bodyweight, aerated lung volumes, viral load in turbinates and lungs, viralshedding in upper respiratory tract, Macroscopic pathologic examinationpost mortem of lung weight, mean percentage of lesion affected lungtissue. Microscopic examination of inflammation parameters of nasalturbinates and lungs. Animal groups 3, 4 and 5 outperformed groups 1 and2 in all macroscopic and in most microscopic parameters tested (data notshown).

Virus Replication in the Upper and Lower Respiratory Tract

On days 0, 1, 2, 3 and 4 after challenge, nose and throat swabs weretaken from the animals under anesthesia. Four days after challenge, theferrets were euthanized by exsanguination under anesthesia after whichfull-body gross-pathology was performed and tissues were collected.Samples of the right nose turbinate and of all lobes of the right lungand the accessory lobe were collected and stored at −80° C. untilfurther processing. Turbinate and lung samples were weighed andsubsequently homogenized with a FastPrep-24 (MP Biomedicals, Eindhoven,The Netherlands) in Hank's balanced salt solution containing 0.5%lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 μg/ml streptomycin,100 U/ml polymyxin B sulfate, 250 μg/ml gentamycin, and 50 U/ml nystatin(ICN Pharmaceuticals, Zoetermeer, The Netherlands) and centrifugedbriefly before dilution.

After collection, nose and throat swabs were stored at −80° C. in thesame medium as used for the processing of the tissue samples.Quadruplicate 10-fold serial dilutions of lung and swab supernatantswere used to determine the virus titers in confluent layers of MDCKcells as described previously (Rimmelzwaan G F et al., J Virol Methods1998 September; 74(1)57-66).

Gross-Pathology and Histopathology

The animals were necropsied according to a standard protocol, aspreviously described (van den Brand J M et al., PLoS One 2012;7(8)e42343). In short, the trachea was clamped off so that the lungswould not deflate upon opening the pleural cavity allowing for anaccurate visual quantification of the areas of affected lung parenchyma.Samples for histological examination of the left lung were taken andstored in 10% neutral-buffered formalin (after slow infusion withformalin), embedded in paraffin, sectioned at 4 μm, and stained withhaematoxylin and eosin (HE) for examination by light microscopy. Sampleswere taken in a standardized way, not guided by changes observed in thegross pathology. Semi-quantitative assessment of influenzavirus-associated inflammation in the lung was performed as describedpreviously (Table 6) (Munster V J et al., Science 2009 Jul. 24;325(5939):481-3). All slides were examined without knowledge of theidentity or treatment of the animals.

Virus Load in Lung and Upper Respiratory Tract Results

All ferrets of control groups 1 (i.n. saline) and 2 (parenteral TIV)showed high titers of replication competent virus in lung (mean titers;5.7 and 5.5 log 10TCID50/gram tissue, respectively) and nasal turbinates(mean titers: 7.2 and 6.9 log 10TCID50/gram tissue, respectively) (Table5). Ferrets of groups 3, 4 and 5 (i.n. Endocine™ adjuvanted splitantigen pH1N1/09 vaccines) had no detectable infectious virus in theirlungs and nasal turbinates. Ferrets of group 6 (i.n. Endocine™adjuvanted whole virus at 15 μg HA) had no detectable infectious virusin their lungs and with a mean titer of 4.1 log 10TCID50/gram tissue asignificant lower virus titer in the nasal turbinates as compared tocontrol group 1 (p=0.02).

Intranasal immunization with Endocine™ adjuvanted pH1N1/09 vaccinesreduced virus titers in swabs taken from the nose and throat as comparedto saline or TIV administration. Virus loads expressed as area under thecurve (AUC) in the time interval of 1-4 dpi, in nasal and throat swabsare shown in Table 5. Virus loads in nasal swabs of groups 3, 4 and 5(i.n. Endocine™ adjuvanted split antigen at 5, 15 and 30 μg HA,respectively), but not of groups 2 and 6 were significant lower than ingroup 1 (group 1 versus groups 3-5; p≦0.03). Virus loads in throat swabsof group 1 and 2 were comparable and significant higher than in groups3, 4, 5 and 6 (p≦0.03).

Gross-Pathology and Histopathology Results

Reduced virus replication in groups intranasally immunized with theEndocine™ adjuvanted pH1N1/09 vaccines corresponded with a reduction ingross-pathological changes of the lungs (Table 5).

The macroscopic post-mortem lung lesions consisted of focal ormultifocal pulmonary consolidation, characterized by well delineatedreddening of the parenchyma. All ferrets in control group 1 (i.n.saline) and group 2 (parenteral TIV) showed affected lung tissue with amean percentage of 50% and 37%, respectively and corresponded with amean relative lung weight (RLW) of 1.5 and 1.3, respectively (Table 5).In contrast, lungs in groups 3, 4, 5 and 6 (i.n. Endocine™ adjuvantedpH1N1/09 vaccines) were much less affected with mean percentages ofaffected lung tissue of 7-8%. The RLWs in these fourEndocine™-vaccinated groups were in line with these observations (in aclose range of 0.8 to 0.9).

The pulmonary consolidation corresponded with an acutebroncho-interstitial pneumonia at microscopic examination. It wascharacterized by the presence of inflammatory cells (mostly macrophagesand neutrophils) within the lumina and walls of alveoli, and swelling orloss of lining pneumocytes. In addition protein rich oedema fluid,fibrin strands and extravasated erythrocytes in alveolar spaces and typeII pneumocyte hyperplasia were generally observed in the more severecases of alveolitis. The histological parameters that were scored aresummarized in Table 5. The most severe alveolar lesions were found inthe control groups 1 (i.n. saline) and 2 (parenteral TIV). Allparameters of alveolar lesions scored lowest in group 5, but in fact thedifferences between the groups 3, 4, 5 and 6 were not significant.

Conclusively, in lungs—The intratracheal challenge with H1N1 influenzaA/Netherlands/602/2009 virus in this ferret model resulted in a slightto severe pneumonia. However, several animals, all from vaccinatedgroups, were not affected by macroscopically discernable lung lesions atall. Based on the macroscopic post-mortem evaluation of lung lesions(estimated % of lung affected), vaccinated (vaccine-A 15 μg HA) group 4and vaccinated (vaccine-A 30 μg HA) group 5 equally suffered the leastlung lesions with both a very low score of 7%, directly followed byvaccinated (vaccine-A 5 μg HA) group 3 and vaccinated (vaccine-B 15 μgHA) group 6 with both 8%. Placebo-PBS-treated group 1 animals sufferedthe most lung lesions with a marked mean score of 50%. Parenterallyvaccinated control group 2 suffered slightly less but still prominentlung lesions with a mean 37%. The mean relative lung weights (RLW) wereevidently in accordance with these estimated percentages of affectedlung tissue, corroborating the validity of these estimated percentagesof affected lung tissue.

The results of the microscopic examination of the lungs confirmed, forthe majority of assessed parameters of lung lesions, the best scores forhighest dosed vaccinated (vaccine-A 30 μg HA) group 5, and a gradualprogression in respiratory lesions correlated to the decrease of HA doseof vaccine-A (groups 3 and 2, respectively). Vaccination with vaccine-B15 μg HA practically equaled the results of lowest dose vaccine-A 5 μgHA (group 3). Placebo-PBS-treatment (group 1) scored by far the worstthroughout all assessed histopathological parameters, closely followedby parenterally vaccinated control group 2. Remarkably, all intranasallyvaccinated animals (groups 3, 4, 5, and 6) were protected from alveolarhaemorrhage.

Overall conclusions—In conclusion therefore, based on the averagedpathology scores in this ferret virus challenge model, the vaccinationwith vaccine-A 30 μg HA (group 5) performed the best and resulted in theleast respiratory laesions, whereas the placebo-PBS-treatment performedthe worst and resulted in the most respiratory lesions. Vaccination withvaccine-A 15 μg HA (group 4) performed just slightly less compared togroup 5, followed by vaccination with vaccine-A 5 μg HA (group 3) thatperformed practically similar compared to vaccination with vaccine-B 15μg HA (group 6). All intranasally vaccinated animals, regardless of thedose and type of vaccine, were protected from alveolar haemorrhage.Parenteral control vaccination (group 2) performed poorly with markedrespiratory lesions and just marginally better compared to theplacebo-PBS-treatment (group 1).

Example 3

The Table 3 below and FIG. 4 compare the vaccine of the presentinvention with other products, FluMist and injectable vaccines in naïveferrets.

TABLE 3 Vaccine Ferrets Vaccine strain Evaluation strain NT titer from(naïve) Dose Route (H1N1) (H1N1) evaluation GSK* N = 6 15 ug HA, IMA/California/7/09 A/The Before (GSK unadjuvanted Netherlands/602/09challenge H1N1) (after 2 vacc) GSK* N = 6 15 ug HA, IM AS03_(A) Novartis# N = 3 15 ug HA, IM A/Brisbane/59/07 (Novartis unadjuvanted TIV)Medimmune # N = 3 1 × 10⁷ IN A/California/7/09 A/California/7/09 Before(pandemic TCID₅₀ (ca) challenge LAIV) (after 2 vacc) GSK ¤ N = 6 15ugHA, SC (GSK TIV) unadjuvanted Eurocine N = 6 15 ug HA, INA/California/7/09 A/The Day 42 Vaccines Endocine ™ Netherlands/602/09(after Immunose ™ 20 mg/ml 2 vacc) FLU ¤ *Baras et al. Vaccine 29 (2011)2120-2126 # Chen et al. JID 2011: 203 ¤ Eurocine Vaccines: the presentstudy

GSK monovalent pandemic vaccine (GSK H1N1), Novartis trivalentinactivated vaccine (Novartis TIV), GSK trivalent inactivated vaccine(GSK TIV) groups had a neutralization titer (NT) titer below 15.

The results show that the vaccine composition of the present invention,Immunose™ FLU, which here means comprising 15 μg HA split influenzaantigen with 20 mg/ml (2%) Endocine™ (group 4, table 2) shows similarneutralizing titers to Medimmune's pandemic LAIV vaccine FluMist (seeFIG. 5) and superior titers to injected vaccines whereas thenon-adjuvanted TIV gives poor response.

Example 4

Evaluation of the humoral immune response in 15 months old mice afterinfluenza vaccination with or without Endocine™

Objective

The objective of the present study was to evaluate theinfluenza-specific antibody response to influenza antigens when combinedwith the Endocine™ adjuvant and delivered intranasally to old (15months) mice.

The Endocine™ adjuvant comprised equimolar amounts of glycerolmonooleate and oleic acid with a final concentration of 20 mg/ml (2%) inthe vaccine composition. In this experiment Immunose™ FLU means non-liveinfluenza antigens mixed with Endocine™.

The influenza-specific antibody response was studied in female micevaccinated with formulations comprising H1N1/California/2009/splitantigen with or without Endocine™, a group receiving saline was includedas control. The mice were vaccinated intranasal on three occasions,separated by three weeks. Blood samples for antibody response evaluationwere collected on day −1, 20, 41 and 63. Experimental groups and vaccinecompositions are illustrated in Table 4.

TABLE 4 Route Dose HA Number Age Vaccination of per Endocine ™ of miceGroup (months) day admin. vaccination (2%) (n) 1 15 0, 21 and 42 in 3μg + 8 Immunose FLU (old) 2 15 0, 21 and 42 in 3 μg − 8 Non-adjuvantedvaccine (old) 3 15 0, 21 and 42 in — − 4 NaCl (old) 4 2 0, 21 and 42 in3 μg + 8 Immunose FLU (young)

Vaccine Preparations and Administration

In this experiment Immunose™ Flu comprises: intranasal drops, 300 μg HA(H1N1/California/2009)/mL+Endocine™ 20 mg/mL (2%). Non-adjuvantedvaccine: intranasal drops containing 300 μg HA(H1N1/California/2009)/mL. NaCl: intranasal drops containing saline 0.9wt %.

Four groups of female Balb/c mice were used in the study. Three groupsinclude mice with an age of 15 months at study initiation (old). Onegroup included mice with an age of 2 months at study initiation (young).Mice were vaccinated intranasal by administration 5 μl of thecomposition to each nostril. The dose of the influenza virus particlesat each immunization was equivalent to 3 μg of hemagglutin (HA), 3 μg HAin 2×5 μl composition. Mice received intranasal vaccinations on threeoccasions, separated by three weeks on day 0, 21 and 42.

Sample Collection and Analysis

Serum samples were collected on day −1, 20, 41 and 63. Samples wereanalysed for specific antibody response, IgG, IgG1, IgG2a and IgA toinactivated split influenza antigens (season 2012/2013 as published bythe WHO, including A/California/07/2009(H1N1)) by ELISA.

The data showed that intranasal administration of Immunose™ Flu to oldmice increased IgG1 titers compared to mice receiving non-adjuvantedvaccine. Further, in old mice vaccinated with Immunose™ Flu an influenzaspecific IgG1 response was detected at day 20 compared to day 41 for oldmice receiving non-adjuvanted vaccine FIG. 6 a. At day 41 IgG2ainfluenza specific antibodies were detected in serum from old micereceiving Immunose™ Flu at a comparable level to young mice receivingImmunose™ Flu, whereas no IgG2a influenza specific antibodies weredetected in old mice receiving non-adjuvanted vaccine FIG. 6 b.Influenza specific IgA titers were only detected in mice vaccinated withImmunose™ Flu, FIG. 6 c.

Collectively the data from this study show that the addition ofEndocine™ to a nasal influenza vaccine increased the influenza-specificIgG and IgG1 titers in serum of old mice when compared to titers inducedby nasal delivery of influenza vaccine without Endocine™. Further, theaddition of Endocine™ was able to induce more IgG2a responders andhigher IgG2a titers after two and three doses in old mice (number ofresponders not shown). The increase in IgG2a titer shows that Immunose™Flu is capable of inducing a Th1 type antibody response. Further, by theaddition of Endocine™ old mice were able to induce an IgA response. Anoverall analysis of end titers of IgG and IgG1 showed a significantdifferences between old and young mice vaccinated with Immunose™ Fludemonstrating that the old mice (15 months at study start) had ahampered immune capacity i.e responded less well to vaccination comparedto young.

Abbreviations Used in Examples:

HA Influenza virus hemagglutinin proteinTCID50 Tissue culture infectious dose 50%PBMC Peripheral blood mononuclear cellsHI Influenza hemagglutination inhibition assay

SOP Standard Operation Procedure

PBS Phosphate buffered salineEDTA Ethylene diamine tetraacetic acidGMT Geometric mean titers (used to express serological data)FCS Fetal Calf Serum (culture medium supplement)VN Virus neutralization assay

DMSO Dimethyl Sulfoxide

TABLE 5 Group^(a) 1 2 3 4 5 6 Clinical score Survival 6/6 5/6 6/6 6/66/6 6/6 Fever 1.7 ± 0.6 1.1 ± 0.4 1.3 ± 0.3 1.2 ± 0.6 1.1 ± 0.6 1.3 ±0.2 (6/6) (6/6) (6/6)  (4/5*) (6/6) (6/6) Body weight loss 18.0 ± 4.6 11.5 ± 2.1  −2.2 ± 2.6  1.7 ± 1.5 2.7 ± 3.3 4.7 ± 3.1 (6/6) (6/6) (1/6)(4/6) (4/6) (6/6) Virology Lung virus load [log₁₀TCID₅₀/g] 5.7 ± 0.5 5.5± 0.9 ≦1.5 (0/6)   ≦1.4 (0/6)   ≦1.3 (0/6)   ≦1.3 (0/6)   (6/6) (6/6)Turbinates virus load 7.2 ± 2.4 6.9 ± 1.5 ≦1.9 (0/6)   ≦1.7 (0/6)   ≦1.7(0/6)   4.1 ± 2.7 [log₁₀TCID₅₀/g] (6/6) (6/6) (3/6) Virus shedding innasal swabs 2.6 (5/6) 1.2 (4/6) 0.058 (1/6)  0.0 (0/6) 0.0 (0/6) 1.4(3/6) Virus shedding in throat swabs  10 (6/6)  10 (6/6) 0.0 (1/6) 0.14(1/6)  0.0 (1/6) 4.2 (5/6) Gross Affected lung tissue [%] 50 ± 25 37 ±21 8 ± 4 7 ± 5 7 ± 5 8 ± 4 (6/6) (6/6) (5/6) (4/6) (4/6) (5/6) pathologyRelative lung weight 1.5 ± 0.5 1.3 ± 0.1 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.20.9 ± 0.1

TABLE 6 Group^(a) 1 2 3 4 5 6 Histopathology Extent of 2.08 ± 0.74 1.88± 0.54 0.42 ± 0.52 0.08 ± 0.20 0.04 ± 0.10 0.42 ± 0.41alveolitis/alveolar (6/6) (6/6) (3/6) (1/6) (1/6) (4/6) damage (score0-3) Severity of alveolitis 2.04 ± 0.68 1.63 ± 0.31 0.50 ± 0.69 0.08 ±0.20 0.04 ± 0.10 0.46 ± 0.46 (score 0-3) (6/6) (6/6) (3/6 (1/6) (1/6)(4/6) Alveolar oedema 29 ± 29 21 ± 19  4 ± 10 0 ± 0 0 ± 0  8 ± 13 (%slides positive) (4/6) (4/6) (1/6) (0/6) (0/6) (2/6) Alveolar 21 ± 40 17± 26 0 ± 0 0 ± 0 0 ± 0 0 ± 0 haemorrhage (2/6) (2/6) (0/6) (0/6) (0/6)(0/6) (% slides positive) Type II pneumocyte 42 ± 34 46 ± 37  8 ± 20  4± 10 0 ± 0  4 ± 10 hyperplasia (4/6) (4/6) (1/6) (1/6) (0/6) (1/6) (%slide positive)

1. A method of immunizing an immune-compromised subject againstinfluenza for reducing virus shedding comprising: administeringintranasally to an immuno-compromised subject a composition comprising:i) one or more non-live influenza virus antigen(s) selected from thegroup consisting of whole inactivated virus, split virus, subunitinfluenza antigen, and recombinant antigens, and ii) an adjuvantcomprising: one or more carboxylic acids, an aqueous medium, and one ormore mono-glycerides. 2-15. (canceled)
 16. The method of claim 1,wherein said immuno-compromised subject is aged ≧65 years.
 17. Themethod of claim 1, wherein said immuno-compromised subject is aprediatric subject.
 18. The method of claim 1, wherein the non-liveinfluenza virus antigen is a split virus antigen.
 19. The method ofclaim 1, wherein the one or more mono-glycerides are selected from thegroup consisting of lauric acid (C12), myristic acid (C14), palmiticacid (C16), palmitoleic acid (C16:1), oleic acid (C18:1), linoleic acid(C18:2), stearic acid, hexanoic acid, caprylic acid, decanoic acid(capric acid), arachidic acid, behenic acid, lignoceric acid,alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid,docosahexaenoic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid,arachidonic acid, erucic acid, and nervonic acid.
 20. The method ofclaim 1, wherein the one or more mono-glycerides are selected from thegroup consisting of palmitoleic acid (C16:1), oleic acid (C18:1), andlinoleic acid (C18:2).
 21. The method of claim 1, wherein the one ormore mono-glyceride is glyceride mono-esterified with oleic acid(glyceryl oleate).
 22. The method of claim 1, wherein the one or morecarboxylic acids are selected from the group consisting of lauric acid,myristic acid, palmitic acid, palmitoleic acid, oleic acid, linoleicacid stearic acid, hexanoic acid, caprylic acid, decanoic acid (capricacid), arachidic acid, behenic acid, lignoceric acid, alpha-linolenicacid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid,gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid,erucic acid, and nervonic acid.
 23. The method of claim 1, wherein theone or more carboxylic acids are selected from the group consisting ofoleic acid and lauric acid.
 24. The method of claim 1, wherein thecarboxylic acid is oleic acid.
 25. The method of claim 1, wherein theadjuvant comprises glyceryl oleate, oleic acid, and an aqeuous medium.26. The method of claim 1, wherein the composition comprisesmonoglycerides in an amount in the range of: about 0.1 g to about 5.0 g,0.1 g to about 2.0 g, about 0.5 g to about 2.0 g, or about 0.5 g toabout 1.5 g per 100 mL of the composition.
 27. The method of claim 1,wherein the composition comprises carboxylic acids in an amount in therange of: about 0.1 g to about 5.0 g, 0.1 g to about 2.0 g, about 0.5 gto about 2.0 g, or about 0.5 g to about 1.5 g per 100 mL of thecomposition.
 28. The method of claim 1, wherein one or moremonoglycerides together with one or more carboxylic acids in an adjuvantmix is at the most: 10% w/v, 5% w/v, 4% w/v, 3% w/v, 2% w/v or 1% w/v ofthe composition.
 29. The method of claim 1, wherein theimmuno-compromised subjects are people aged ≧65 years, pregnant women,premature babies, cancer patients, persons receiving chemotherapy,persons receiving radiation therapy, organ transplant patients, personsundergoing solid organ transplants, stem cell transplant patients,persons undergoing hematopietic allogenic stem cell transplantation,persons undergoing hematopoietic autologous stem cell transplantation,HIV infected patients, persons with AIDS, patients withgraft-versus-host disease, patients on immune suppressive drugs,patients receiving glucocorticoid therapy or patients receiving steroidtherapy, persons with chronic diseases, patients with end stage endstage renal disease, diabetes, or cirrhosis.